System and method for deforming, imaging and analyzing particles

ABSTRACT

A system for deforming and analyzing particles includes a substrate defining an inlet, and an outlet; a fluidic pathway fluidly coupled to the inlet and the outlet and defining a delivery region upstream of a deformation region configured to deform particles, wherein the fluidic pathway comprises a first branch configured to generate a first flow, and a second branch configured to generate a second flow that opposes the first flow, wherein an intersection of the first flow and the second flow defines the deformation region; a detection module including a sensor configured to generate a morphology dataset characterizing deformation of the particles, and a photodetector configured to generate a fluorescence dataset characterizing fluorescence of the particles; and a processor configured to output an analysis of the plurality of particles based at least in part on the deformation dataset and the fluorescent dataset for the plurality of particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims priority to U.S. Provisional Patent ApplicationNo. 61/718,077 filed on Oct. 24, 2012, U.S. Provisional PatentApplication No. 61/718,092 filed on Oct. 24, 2012, and U.S. ProvisionalPatent Application No. 61/719,171 filed on Oct. 26, 2012. Priority isclaimed pursuant to 35 U.S.C. §119. The above-noted Patent Applicationsare incorporated by reference as if set forth fully herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under 1150588, awardedby the National Science Foundation and N66001-11-1-4125, awarded by theSpace and Naval Warfare Systems Command. The Government has certainrights in the invention.

TECHNICAL FIELD

This invention relates generally to the cytometer field, and morespecifically to an improved system and method for deforming andanalyzing particles such as cells in the cytometer field.

BACKGROUND

There is growing evidence that cell deformability is a useful indicatorof abnormal cytoskeletal changes, and may provide a label-free biomarkerfor determining cell states or properties, such as metastatic potential,cell cycle stage, degree of differentiation, and leukocyte activation.Clinically, a measure of metastatic potential could guide treatmentdecisions, or a measure of degree of differentiation could preventtransplantation of undifferentiated tumorigenic stem cells inregenerative therapies. For drug discovery and personalized medicine, ameasure of cytoskeletal integrity could allow screening forcytoskeletal-acting drugs or evaluation of cytoskeletal drug resistancein biopsied samples. Cell deformability can further provide insight intomechanotransduction pathways for different cell lines, opening newavenues of discovery in cellular biomechanics. Currently, implementationof these techniques and analyses is cost-prohibitive andlabor-intensive, which is a substantial limiting factor in clinical andresearch applications. Current platforms for cell deformation techniquesand analyses suffer from a large number of limitations, including one ormore of the following: limited throughput, inconsistency, limitedcharacterization of sample heterogeneity, speed, and labor intensity. Inparticular, platforms optimized for biophysics research operate at ratesof approximately 1 cell/minute, which significantly hampers one'sability to process and analyze a large number of heterogeneousparticles.

Thus, there is a need in the cytometer field to create a new andimproved system and method for deforming and analyzing particles. Thisinvention provides such a new and improved system and method.

SUMMARY

In one embodiment, a system for deforming and analyzing a plurality ofparticles carried in a sample volume includes a substrate defining aninlet, configured to receive the sample volume, and an outlet; a fluidicpathway fluidly coupled to the inlet and the outlet and defining adelivery region located upstream of a deformation region configured todeform one or more particles in the plurality of particles, wherein thefluidic pathway includes a first branch configured to deliver a firstportion of the sample volume in a first flow, and a second branchconfigured to deliver a second portion of the sample volume in a secondflow that opposes the first flow, wherein an intersection of the firstflow and the second flow defines the deformation region, and wherein thedelivery region is fluidically coupled with at least one of the firstand the second branches; a detection module comprising a sensorconfigured to generate a morphology dataset characterizing deformationof one or more particles in the plurality of particles, and comprising aphotodetector configured to generate a fluorescence datasetcharacterizing fluorescence of one or more particles in the plurality ofparticles; and a processor configured to output an analysis of theplurality of particles based at least in part on the morphology datasetand the fluorescent dataset for the plurality of particles.

In another embodiment, a system for deforming and analyzing a pluralityof particles carried in a sample volume, the system includes a substratedefining an inlet and an outlet and a fluidic pathway interposed therebetween; a focusing region disposed in the fluidic pathway and coupledat a downstream end thereof to a trifurcation comprising a centralbranch, a first side branch, and a second side branch; a deformationregion disposed downstream of the trifurcation comprising anintersection formed between the central branch, the first side branch,and the second side branch, wherein first side branch and the secondside branch intersect with the central branch is a substantiallyorthogonal orientation; a detection module comprising a sensorconfigured to generate a morphology dataset characterizing deformationof one or more particles in the plurality of particles, and comprising aphotodetector configured to generate a fluorescence datasetcharacterizing fluorescence of one or more particles in the plurality ofparticles; and a processor configured to output an analysis of theplurality of particles based at least in part on the deformation datasetand the fluorescent dataset for the plurality of particles.

In another embodiment, a method for deforming and analyzing a pluralityof particles carried in a sample volume, the method includes: receivingthe sample volume comprising the plurality of particles; diverting afirst portion of the sample volume in a first flow and a second portionof the sample volume in a second flow, substantially opposed to thefirst flow, such that an intersection of the first and the second flowsdefines a deformation region; delivering the plurality of particles intothe deformation region; generating a morphology dataset characterizingdeformation of one or more particles of the plurality of particleswithin the deformation region; generating a fluorescence datasetcharacterizing fluorescence of one or more particles of the plurality ofparticles within the deformation region; and outputting an analysis ofthe plurality of particles based at least in part on the deformationdataset and the fluorescent dataset for the plurality of particles.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of an embodiment of a system fordeforming and analyzing particles;

FIGS. 2A and 2B are schematic representations of an embodiment of aportion of a system for deforming and analyzing particles;

FIGS. 3A and 3B depict a variation of a delivery region in an embodimentof a system for deforming and analyzing particles;

FIGS. 4A and 4B depict a variation of a delivery region in an embodimentof a system for deforming and analyzing particles;

FIG. 5 depicts a variation of a deformation region in an embodiment of asystem for deforming and analyzing particles;

FIG. 6A-6C depict variations of a deformation region in an embodiment ofa system for deforming and analyzing particles;

FIG. 7 depicts an example of a fluidic pathway in an embodiment of asystem for deforming and analyzing particles;

FIG. 8A depicts an example of a fluidic pathway in an embodiment of asystem for deforming and analyzing particles;

FIG. 8B depicts another example of a fluidic pathway in an embodiment ofa system for deforming and analyzing particles;

FIG. 9A depicts an alternative example of a deformation region in anembodiment of a system for deforming and analyzing particles;

FIG. 9B depicts an example of a fluidic pathway in an embodiment of asystem for deforming and analyzing particles using the deformationregion illustrated in FIG. 9A;

FIG. 9C illustrates the fluidic pathway of FIG. 9B with the resistanceslabeled for various branch and inlet channels;

FIG. 9D illustrates an embodiment of a fluidic pathway that combinesoff-axis squeezing at a first deformation region followed by a secondarydeformation region in which particles are subject to deformation at anintersection of opposing flows;

FIG. 9E illustrates a simplified resistor diagram of the combined HA-DCdevice of FIG. 9D.

FIG. 9F illustrates another embodiment of a fluidic pathway in whichhydropipette aspiration is combined with rapid inertial solutionexchange for integrated sample preparation and analysis.

FIG. 9G illustrates series of magnified images of selected regions ofthe device of FIG. 9F.

FIGS. 10A-10C depict variations of a detection module in an embodimentof a system for deforming and analyzing particles;

FIG. 11A-11C depict alternative variations of a detection module in anembodiment of a system for deforming and analyzing particles;

FIGS. 12A-12C depict alternative variations of a detection module in anembodiment of a system for deforming and analyzing particles;

FIG. 13A-13C depict example particle characteristics extracted using anembodiment of a system for deforming and analyzing particles;

FIG. 14 depicts an example synchronization method for an embodiment of asystem for deforming and analyzing particles;

FIG. 15 is a flowchart of an embodiment of a method for deforming andanalyzing particles; and

FIG. 16 is a flowchart of an embodiment of a method for deforming andanalyzing particles.

DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

The following descriptions of the illustrated embodiments of theinvention are not intended to limit the invention to this preferredembodiment, but rather to enable any person skilled in the art of flowcytometers to make and use this invention.

1. System

As shown in FIG. 1, a system 100 according to one embodiment isdisclosed for deforming and analyzing a plurality of particles carriedin a sample fluid. As used herein, the terms “particle” or “particles”are meant to encompass small objects that can be contained within fluidflow. A particle may include a biological object such as a cell or evenan organelle. According to this embodiment, the system 100 includes asubstrate 110 defining an inlet 104 and an outlet 106; a fluidic pathway120 fluidly coupled to the inlet 104 and the outlet 106 and defining adelivery region 130 located upstream of a deformation region 140configured to deform one or more particles that enter the deformationregion 140; a detection module 150 including a sensor 155 configured togenerate data characterizing the deformation of one or more particlescontained within a plurality of particles flowing through the system 100and a photodetector 160 configured to generate data characterizingfluorescence of each particle in the plurality of particles; and aprocessor 180 configured to generate an analysis based upon deformationand fluorescence of the one or more particles.

The system 100 functions to enable the deformation of single particlesin a high-throughput and consistent manner, with the ability tosimultaneously generate and analyze multiple data types characterizingthe single particles. Preferably, the system 100 further functions toenable the generation of data that directly correlates surfacebiomarkers of phenotype with mechanical properties at thesingle-particle level. This can allow the generation of a directquantitative comparison between biomolecular properties and mechanicalproperties. Preferably, the system 100 is used to process and analyzebiological particles, such as cells, and in specific applications, thesystem 100 can be used to analyze leukocyte activation, stem celldifferentiation, cellular response to drugs, and cancer cell malignancyby way of correlating cellular deformation with biomolecular phenotypesusing fluorescence assays. Besides correlating to biomolecularphenotypes, combining biomolecular and deformability-based data canprovide additional classification accuracy. However, the system 100 canalternatively be used to process, deform, and analyze any other suitablebiological particle or non-biological particles.

1.1 System—Substrate

The substrate 110 functions to provide a platform by which particles ofinterest can be deformed and analyzed. The substrate preferablycomprises microfluidic elements that enable deformation of the particlesof interest, and facilitates data generation from the deformed particlesof interest by defining a suitable configuration of the microfluidicelements relative to other elements of the system 100 (e.g., pump,detection module, waste chamber). In one variation, the microfluidicelements of the substrate include an inlet 104 and an outlet 106 forreceiving a sample volume and transmitting a processed sample volume,respectively, from the substrate 110. In a first specific example, asshown in FIGS. 2A and 2B, the substrate 110 includes a single inlet 104defined at a first surface of one end of the substrate 110 and twooutlets 106 defined at an opposite end of the substrate 110. However,other variations of the substrate 110 can comprise any other suitableelement(s) in any suitable configuration that facilitates coupling withelements external to the substrate 110 for deforming, processing, andanalyzing a sample volume containing particles of interest. For example,the substrate 100 may include multiple inlets 104 and multiple outlets106. The inlet(s) 104 and outlet(s) 106 of the substrate 110 can bedefined at any suitable end, at any suitable surface, and/or within anysuitable region of the substrate 110. Furthermore, an inlet 104 can beconfigured to receive any suitable processing fluid (e.g., sheath fluid,reagent, buffer, wash, etc.) to facilitate sample processing.

In some variations, the substrate 110 can be configured to be a reusableelement and in other variations, the substrate 110 can be configured tobe a disposable element. In variations wherein the substrate 110 isreusable, the substrate 110 can be configured to couple to a module forwashing or flushing the substrate 110 (e.g., through the inlet oroutlet) after uses of the substrate. Alternatively, in these variationsof a reusable substrate 110, the substrate 110 can be configured to beself-cleaning or self-washing (e.g., using surface coatings, bygeometric configuration of fluidic pathways, etc.). In other variations,the substrate can be configured to be reusable for a certain number ofuses or until failure (e.g., failure by clogging), and then disposed tobe replaced. In any of these variations, the substrate 110 can comprisealigners (e.g., slots, pins, guides, etc.) configured to facilitatealignment of the substrate 110 within the system 100 and relatively toother elements of the system 100. The substrate 110 may be a monolithicsubstrate or the substrate 110 may be formed from multiple layers thatare bonded or otherwise secured to one another to form the appropriatemicrofluidic elements within the substrate 110.

The inlet 104 functions to receive a sample volume, including aplurality of particles of interest, to initiate processing and analysisof the particles within the substrate 110. Preferably, the inlet 104 isconfigured to receive the sample volume and the plurality of particlesfrom a fluid delivery module including a pump 112, as shown in FIG. 1;however, the inlet can be configured to receive the sample volume in anyother suitable manner. In other variations, the pump 112 can be asyringe pump containing the sample volume and the plurality ofparticles, or any other fluid pump configured to provide at least one ofa positive pressure and a negative pressure, in order to deliver thesample volume and the plurality of particles into the inlet 104.Additionally, the pump 112 can be manually or automatically operated,but is preferably configured to transmit the sample volume into theinlet 104 at a uniform flow rate that can be adjusted. Furthermore, thepump 112 can be coupled to any suitable conduit (e.g., tubing, conduit,manifold) configured to transmit the sample volume (e.g., from a samplewell coupled to the substrate) into the inlet 104, and can comprise avalve and/or a pressure sensor in order to control and detect flowparameters. In one specific example, the pump 112 is automaticallycontrolled and configured to provide an adjustable flow rate thatenables particle focusing and achieves a desired particle deformation.Alternatively, the pump 112 may be controlled to achieve a particularparticle throughput. Furthermore, in still other alternative examples,the pump 112 may be configured to deliver a sample volume includingcells (i.e., particles of interest) with a density between 200,000cells/mL and 8 million cells/mL.

In specific applications with biological particles, the plurality ofparticles (e.g., cells) can be prepared for fluorescence-based assaysprior to delivery into the inlet 104 of the substrate 110. Preferably,the plurality of particles is prepared using an approach that omitsfixation, which can affect deformation of the particles in unknownand/or unpredictable ways. The cells are preferably labeled with atleast one fluorescently-labeled biochemical probe (e.g., SSEA4 probe,Oct4 probe, TRA-1-60 probe, CD34 probe, CD38 probe, HLA-DR probe, CD64probe, etc.) bound to cell surface proteins or other biomarkers, whichfacilitates identification of biomolecular markers that can be extractedas fluorescence data. The cells can additionally be processed withcell-permeable stains to facilitate identification. However, theplurality of particles can be processed in any other suitable mannerprior to delivery into an inlet 104, and/or during transmission throughany element of the system 100 (e.g., fluidic pathway, etc.).

Preferably, the inlet 104 is configured to form a hermetic seal aboutthe fluid delivery module and/or the pump 112, such that the samplevolume does not leak from the inlet 104; furthermore, the inlet 104 ispreferably configured to be reversibly coupled to the fluid deliverymodule and/or the pump 112. However, the inlet 104 can be configured tocouple to the fluid delivery module and/or the pump 112 in any othersuitable manner. In one variation, the inlet 104 is configured to coupleto the pump 112 by a threaded male-female coupling configured to producea hermetic seal. In another variation, the inlet 104 can additionally oralternatively comprise an o-ring configured to facilitate generation ofthe hermetic seal. In still other variations, the inlet 104 canadditionally or alternatively comprise any other suitable sealant (e.g.,resealable septum, silicone sealant, sealing putty) for generation ofthe hermetic seal.

The outlet 106 functions to transmit the sample volume including theplurality of particles of interest from the substrate 110, after thesample volume has been processed. Preferably, the outlet 106 isconfigured to transmit the processed sample volume as waste from thesubstrate 110; however, the outlet 106 can alternatively be configuredto transmit the processed sample volume from the substrate 110 forfurther processing and analysis. In one variation, the outlet 106 can beconfigured to couple to a waste chamber as seen in FIG. 2A that isconfigured to receive waste fluids from the outlet 106. In thisvariation, the waste chamber can be integrated (e.g., of unitaryconstruction, physically coextensive) with the substrate 110, such thatthe outlet 106 is configured to deliver waste fluids into the wastechamber of the substrate 110. In another variation, the outlet 106 canbe configured to couple to a fluid conduit that delivers the processedsample volume to another module for further processing. Similar to theinlet 104, the outlet 106 is preferably configured to form a hermeticseal about a point of coupling (e.g., to a waste chamber, to a modulefor further processing), and can comprise any one or more of: amale-female threaded coupling, an o-ring, septum, and a sealant thatfacilitates generation of the hermetic seal. In other variations,however, the outlet 106 can be configured to couple to any othersuitable element in any other suitable manner, for example, using one ormore microfluidic conduits or channels.

The substrate 110 is preferably composed of an optically transparentmaterial with no autofluorescence, in order to facilitate detection ofsample particle characteristics (e.g., deformation characteristics,mechanical properties, fluorescence characteristics) without opticalinterference from the substrate 110. However, the substrate 110 can besufficiently transparent and/or composed of a material with sufficientlylow autofluorescence in order to enable detection of particlecharacteristics. Additionally, the substrate 110 can comprise anystructures or elements configured to reflect light toward particlespassing through the substrate 110, in order to enhance detection ofparticle characteristics and parameters by a detection module 150.Furthermore, the substrate 110 can include any suitable structure(s) formicrofluidic applications, including glass structures, polymericstructures, or composite structures. In one variation, the substrate 110can be composed of a polymeric material that is processable to form theinlet(s) 104, the outlet(s) 106, and/or any other suitable element(s) ofthe substrate 110. In a specific example of this variation, thesubstrate 110 is composed of polydimethylsiloxane (PDMS) contained on aoptically transparent solid surface such as glass, with inlet(s) 104,outlet(s) 106, and microfluidic elements defined by a lithographicprocess (e.g., photolithography), such as a process described in U.S.Pub. No. 2013/0177935, entitled “Method and Device for High ThroughputCell Deformability Measurements”, which is incorporated herein in itsentirety by this reference. In other variations of this example,substrate features can be additionally or alternatively defined by anyother suitable process (e.g., micromachining, molding, etching, 3Dprinting, etc.). Alternatively, the substrate 110 can comprise or becomposed of any other suitable material, processable by any othersuitable method to form features of the substrate 110 (e.g., inlets,outlets, fluidic pathways, etc.).

1.2 System—Fluidic Pathway

The fluidic pathway 120, as shown in FIGS. 1 and 2A-2B, is preferablyfluidically coupled to the inlet(s) 104 and the outlet(s) 106 of thesubstrate 110, and functions to facilitate focusing and deformation ofthe plurality of particles of the sample volume. The fluidic pathway 120is also preferably configured between the inlet(s) 104 and the outlet(s)106, such that any pressure differential (e.g., generated by the pump112) along the fluidic pathway 120 facilitates fluid flow through atleast a portion of the fluidic pathway 120. Preferably, the fluidicpathway 120 is at least partially defined within the interior of thesubstrate 110 (e.g., by a lithographic process, by etching, bymicromachining, by 3D printing, etc.); however, the fluidic pathway 120can be partially or completely defined external to the substrate 110.Preferably, the fluidic pathway 120 comprises a delivery region 130 thatis located upstream of a deformation region 140, such that the pluralityof particles of the sample volume can be transmitted from an inlet 104,focused within the delivery region 130, and transmitted to thedeformation region 140 for deformation and analysis. In thisconfiguration, the delivery region 130 is interposed between theinlet(s) 104 and the deformation region 140.

The delivery region 130 functions to focus at least a subset of theplurality of particles into the deformation region 140 along a commonequilibrium point or streamline, such that each particle in theplurality of particles experiences sufficiently uniform flow anddeformation conditions in a manner that limits experimental variability.Additionally, the delivery region 130 is preferably configured tocooperate with conditions provided by the pump 112, such that theplurality of particles flows in single file at a substantially uniformvelocity (e.g., with particle size-dependent fluctuations in velocity in5-10% range) into the deformation region 140. Alternatively, thedelivery region 130 and the pump 112 can be configured to transmit theplurality of particles in non-single file, and/or with any suitablevelocity profile (e.g., variable velocity profile) into the deformationregion 140. Preferably, the delivery region 130 provides inertialfocusing and can comprise at least one curved confined channel 132configured to provide inertial focusing of the plurality of particlesinto the deformation region 140. In a first variation of the deliveryregion 130′, an example of which is shown in FIG. 3A, the curved channel132 can be characterized by a profile described, for example, in D. R.Gossett et al., “Particle focusing mechanisms in curving confinedflows,” Analytical Chemistry, 81, 8459 (2009), which is incorporatedherein in its entirety by this reference. The curved channels 132 may besymmetric or asymmetric although asymmetric curved channels 132 aregenerally preferred. Furthermore, in this variation, the delivery region130 can comprise multiple curved confined channels 132 coupled inseries, as shown in FIG. 3A, that enable focusing of particles into thedeformation region 140. In the first variation of the delivery region130, the curved channel 132 configuration focuses the plurality ofparticles along a single projected line, with each particle positionedwithin one of two focal planes, as shown in FIG. 3B. While thisembodiment focuses particles at two focal planes as seen in FIG. 3B,particles at both locations can be imaged using a single detectionmodule 150 that operates at a relatively low magnification. At highermagnifications, image processing may be needed to extract images at thetwo focal planes for deformation analysis.

In a second variation of the delivery region 130″, as shown in FIGS. 4Aand 4B, the delivery region comprises a straight channel 133 that isinterspersed with a plurality of serially arrayed constrictions inheight 134, orthogonally arranged relative to the flow direction thatprovides focusing based upon inertial focusing and geometry-inducedsecondary flows. The straight channel 133 in the second variation ispreferably defined by a low aspect ratio (defined as height divided bywidth), and the combination of inertial focusing upstream and a pair oflocal helical secondary flows induced by the height constrictions 134provides focusing of each particle in the plurality of particles, insequence, to a single position. In this variation of the delivery region130″, at a finite Reynolds number (Re), particle migration in thestraight channel 133 occurs due to a balance of two inertial liftforces: shear-gradient (FSL) and wall-effect (FWL) lift forces. Aninteraction between a particle wake and a wall of a channel of thedelivery region 130 produces a FWL directed toward the channelcenterline, while a parabolic velocity profile causes a shear-gradientinduced FSL directed toward a channel wall throughout the channel,except where it is zero at the channel centerline; the balance of theFSL and FWL forces thus leads to well-defined equilibrium particlepositions (e.g., along centerlines of channel walls for a channel with arectangular cross section, as in FIG. 4B. Then, the plurality of heightconstrictions 134 induce a pair of helical secondary flows configured toinduce lateral motions that compete with the inertial lift forces todirect the plurality of particles into a single particle position on achannel wall opposite to the plurality of height constrictions 134, asshown in FIGS. 4A and 4B.

In a specific example of the second variation of the delivery region130, the straight channel 133 is a rectangular channel with an aspectratio of approximately 0.5 with a width of84 micrometers, a height of41.5 micrometers, and a length of 6 cm. In the specific example, thedelivery region comprises thirty (30) constrictions in height that are21 micrometers in height, 40 micrometers in length, and spaced apart by1 mm. It should be understood that the particular dimensions discussedabove should be regarded as exemplary as other dimensions for thechannel and the constrictions may be used. Further, as disclosed herein,a different number of constrictions (e.g., fewer than thirty (30)) maybe used to focus the plurality of particles. Prior to entering a heightconstriction 134, the plurality of particles are focused alongcenterlines proximal to each of two to four walls of the straightchannel 133, depending on aspect ratio. Then, after successivelyentering each height constriction 134 in the plurality of heightconstrictions 134, the particles of the plurality of particles deviatetoward a single equilibrium position based upon a balance between strongFSL forces and weaker FWL forces. In the specific example, focusing to asingle stream defining a single equilibrium position achieved a focusingefficiency (i.e., percentage of particles reaching the equilibriumposition) of 99.77% after the plurality of particles enteredapproximately twenty-five (25) height constrictions of the plurality ofheight constrictions 134. The height constrictions 134 may projectupward from a lower base or, alternatively, project downward from anupper surface. Furthermore, the full width at half maximum (FWHM)defining focusing tightness was 10.995 micrometers in the deliveryregion for 10 micrometer diameter particles, indicating sufficientlynarrow particle focusing. Additionally, focusing in the specific exampleof the second variation improved with Re, such that at Re=83.33, allparticles in the plurality of particles were focused at a singleequilibrium position, facilitating measurements by a detection module150 (e.g., a module defining a single focal depth). In alternatives tothe second variation, the straight channel 133 can be replaced by acurved channel 132, such as a curved channel described in the firstvariation of the delivery region 130 described above. Variations using acurved channel 132 can decrease a total channel length used for thedelivery region 130.

In alternative variations, the delivery region 130 can be configured forany one or more of the following types of focusing: hydrodynamicfocusing, focusing using a sheath fluid, dielectrophoretic focusing,ultrasonic focusing, magnetic focusing, and any other suitable focusingmethod. In one example, the delivery region 130 can be configured todirect the plurality of particles into a branch of the fluidic pathway120 along a common streamline, and simultaneously, to direct portions ofthe sample volume not including the plurality of particles into otherbranches of the fluidic pathway 120. As such, the delivery region 130can be used to separate the plurality of particles from the samplevolume, and to utilize a portion of the sample volume for a subsequentuse. For example, one subsequent use of a sample volume that does notcontain particles includes using the diverted sample volume to squeezeparticles. This can be seen, for example, in the trifurcation structureof FIG. 7 whereby two branches 121′, 123′ divert fluid that is free ofparticles that is later used in a deformation region 140. Furthermore,the delivery region 130 is preferably configured to direct the pluralityof particles along a centerline of a channel of the fluidic pathway 120to facilitate measurements by a detection module 150; however, thedelivery region 130 can be additionally or alternatively be configuredto direct the plurality of particles along any suitable portion (e.g.,centerline, periphery) of a channel of the fluidic pathway 120 or abranch of the fluidic pathway 120, in order to divert the plurality ofparticles into specific regions for processing.

The deformation region 140 functions to deform one or more of theplurality of particles by using opposing flows, according to oneembodiment, as shown in FIG. 5. In this embodiment, the deformationregion 140 is formed at an intersection of opposing flows, whereby aparticle entering the intersection of the opposing flows undergoesdeceleration and is compressed by the opposing flows, leading tocompression of a particle along one axis and extension of each particlealong another axis. However, alternative variations of the deformationregion can mechanically deform the plurality of particles using anyother suitable mechanism. In the embodiment of FIG. 5, the opposingflows are substantially coaxially aligned and flow anti-parallel to eachother; however, the opposing flows can be unaligned and/or not flow inanti-parallel directions. In the embodiment of FIG. 5, the particlesenter from only one side of the extension region 140. The opposing flowenters the extension region 140 but is free of particles. Preferably, afirst flow and a second flow in the opposing flows are generated fromthe sample volume (i.e., in a self-sheathing manner), such that a firstportion of the sample volume is used to generate the first flow and asecond portion of the sample volume is used to generated the second flowthat opposes the first flow. This can be achieved at branches of thefluidic pathway 120 that are configured to diverge and/or converge(e.g., by way of bifurcations, trifurcations, etc.). This is seen, forexample, in the embodiments of FIGS. 1. 2A, 7, 8A, 8B, 9B, 9C, and 9D.

Preferably, the deformation region 140, in cooperation with flowconditions provided by the pump and the delivery region 140, generates asuitable amount of deformation that is substantially uniform across theplurality of particles that have the same mechanical characteristics anddoes not result in saturation of measurements. For instance, a low flowrate generated by the pump can result in non-uniform deformation at thedeformation region 140, and a high flow rate generated by the pump canresult in particles being deformed beyond an imaging window and/orparticle lysis, leading to measurement saturation. The flow rate(s) usedto deform the plurality of particles at the deformation region 140 ispreferably associated with a cross-sectional dimension (e.g., diameter,width) of at least a portion of the fluidic pathway 120 (e.g., branch,delivery region, deformation region), with higher flow rates requiredfor larger cross-sections. In one variation, the flow conditionsprovided by the pump can be governed based upon an analysis of channelresistances (e.g., a ratio of resistances between flow branches), whichat least partially depend upon a cross-sectional dimension. In examplesof this variation, as shown in FIGS. 6A-6C, a first flow and a secondflow in the opposing flows are designed to have a ratio of resistancesthat generates a suitable opposing flow profile, while maintaining asufficient number of particles (e.g., 95% of the plurality of particles)within one of the first flow and the second flow. For example, withrespect to FIG. 6A, the first flow may include substantially all of theplurality of particles while the second, opposing flow is substantiallyfree of particles. In other examples, a first flow and a second flow inthe opposing flows can have matched or unmatched resistances, in orderto generate a desired deformation of each particle in the plurality ofparticles. For example, with reference to FIG. 6B, the resistance ofR_(outlet 2) may be larger than the resistance of R_(outlet 1) in whichcase a larger percentage of particles will exit the deformation region140 via R_(outlet 1).

In one embodiment, the deformation region 140 receives the plurality ofparticles from only one flow in the opposing flows that enter thedeformation region 140, such that a first flow provides the focusedplurality of particles (i.e., from the delivery region 130) and at leastone other flow opposes the first flow at an intersection to generate thedeformation region 140. The plurality of particles is thus configured toenter the deformation region 140 from a single direction. Thesingle-direction design aspect is important when used in conjunctionwith fluorescent detection because fluorescent measurements can be madein a single location upstream of the deformation region 140 where thevelocity of entering particles is substantially uniform. However, theplurality of particles can alternatively be divided into multiple flowsof the opposing flows, and configured to enter an intersection of theopposing flows (i.e., a deformation region 140) from at least twodirections for deformation. In variations wherein the plurality ofparticles is divided into multiple flows, the multiple flows eachpreferably comprise a delivery region 130 to focus particles alongcommon streamlines prior to deformation. However, any portion of themultiple flows can omit a delivery region 130 in other variations.

In one variation, the fluidic pathway 120 comprises a first branchconfigured to deliver a first portion of the sample volume in a firstflow, and a second portion of the sample volume in a second flow, suchthat sample volume is divided into at least two flows that cooperate tofocus and deform the plurality of particles. In a first example of thisvariation, as shown in FIG. 7, the fluidic pathway 120′ includes atrifurcation 125′ that divides the sample volume into a first branch121′ in a first flow, a second branch 122′ in a second flow, and a thirdbranch 123′ in a third flow. In the first example, the delivery region130′ is coupled to the first branch 121′, the second branch 122′, andthe third branch 123′ of the trifurcation 125′, in a manner that focusessubstantially all of the plurality of particles into the second branch122′ of the trifurcation. Additionally, in the first example, the firstand the third flows are substantially devoid of particles of theplurality of particles, and the first and the third branches 121′, 123′are configured to direct the first and the third flows, respectively, ina direction that opposes the second flow of the second branch 122′(illustrated by arrow A in FIG. 7). In the first example, theintersection of the first, the second, and the third flows at a point ofopposition, forms the deformation region 140 for deformation of theplurality of particles. Furthermore, in the first example, thedeformation region 140 is configured to couple to a first outlet 106 anda second outlet 106′, for transmission of processed sample fluid out ofthe substrate 110. In variations of the first example, the deliveryregion 130′ can be configured to divert the plurality of particles intoany one or more of the first, the second, and the third branches 121′,122, 123′, and the fluidic pathway 120′ can be configured to couple toany suitable number of inlets 104 and outlets 106 for reception of thesample volume (or other fluids) and transmission of fluids from thesubstrate 110.

FIG. 6C schematically illustrates the fluidic resistances of thetrifurcation embodiment of FIG. 7. R1 represents the fluidic resistancein the second branch 122′. R2 represents the fluidic resistance in thereturn sheath flow entering the deformation region 140. R3 representsthe fluidic resistance in the first and third branches 121′, 123′. Inthis embodiment, as part of the design criteria, R1=R3/2+R2.

In another embodiment, as shown in FIG. 8A, the fluidic pathway 120″includes a bifurcation 124″ that divides the sample volume into a firstbranch 121″ in a first flow and a second branch 122″ in a second flow,wherein the first flow and the second flow each contain a subset of theplurality of particles of the sample volume. In the second example, thedelivery region 130″ is coupled to the first branch 121″ downstream ofthe bifurcation 124″, and a second delivery region 131″ is coupled tothe second branch 122′ downstream of the bifurcation 124″, such that thesubsets of the plurality of particles are focused within the deliveryregion 130″ and the second delivery region 131″. Furthermore, in thesecond example the first branch 121″ and the second branch 122″ areconfigured to direct the first flow and the second flow, respectively,in opposing directions downstream of the delivery regions 130″, 131″,such that an intersection of the first flow and the second flow definesthe deformation region 140″. The deformation region 140″ in the secondexample is configured to couple to a first outlet 106 and a secondoutlet 106′, for transmission of processed sample fluid out of thesubstrate 110. In one alternative embodiment, the fluidic pathway 120″can be configured to divert a first portion of the sample volume (e.g.,by inertial focusing, by using multiple inlets), with substantially allparticles of the plurality of particles, into the first branch 121″,such that the second branch 122″ does not receive any particle of theplurality of particles in the second flow (or vice versa). In thisvariation of the second example, the second delivery region 131″ can beomitted, such that the first branch is configured to focus the pluralityof particles into the deformation region 140″ formed at the intersectionof the first and the second branches 121″, 122″. In this variation ofthe second example, the plurality of particles is thus configured toenter the deformation region 140 from a single direction.

FIG. 8B illustrates the alternative embodiment discussed above whereinsubstantially all particles of the plurality of particles are divertedinto the first branch 121′″ while the second branch 122′″ issubstantially free of particles. In this example, the bifurcation 124′initiates from a curved portion of an upstream focusing region 127whereby the particles are preferentially aligned along fluid streamlinesthat are shunted to the first branch 121′″. The particles then passthrough a delivery region 130′″ prior to entering an imaging region 129located immediately upstream of a deformation region 140′. Particlesleave the deformation region 140′″ via one or both outlets 106″, 106′″.A filter 190 is illustrated coupled to the upstream of the focusingregion 127.

Furthermore, in alternative variations, each particle in the pluralityof particles can be deformed by an opposing flow that has a directioncomponent that is transverse to a prevailing direction of the flowcontaining the particles. In these alternative variations, at least oneopposing flow can be generated with or without using any portion of thesample volume (e.g., by an outside flow that is injected or pumped togenerate an opposing flow). In one alternative variation, an opposingflow that is coaxially aligned with, but anti-parallel to a flowcontaining at least a portion of the plurality of particles, can begenerated by an outside flow that is transmitted through an inlet. Inanother alternative variation, at least one opposing flow can begenerated in a direction not coaxially aligned with a flow containing atleast a portion of the plurality of particles, such that the opposingflow has a direction component that is transverse to a prevailingdirection of the flow containing the particles. In this alternativevariation, the opposing flow is preferably substantially orthogonal to aprevailing direction of the flow containing the particles; however, theopposing flow can alternatively be non-orthogonal to and non-parallel tothe flow containing the particles.

In one example of an alternative variation, as shown in FIG. 9A, a firstflow containing the plurality of particles is configured to enter thedeformation region 140 along a first direction via central branchchannel 122″, after being focused in an embodiment of the deliveryregion 130 described above. A first inlet 135 and a second inlet 135′ atthe deformation region 140 are configured to provide a first opposingflow and a second opposing flow that are anti-parallel (i.e., off-axis)to the first opposing flow. In one embodiment, the first opposing flowand the second opposing flow are both substantially orthogonal to thefirst flow containing the plurality of particles. The first opposingflow and the second opposing flow in this example are equal andopposite; however, the first opposing flow and the second opposing flowcan alternatively be non-equal and/or non-opposite in variations of thisexample. In addition, while the first inlet 135 and the second inlet135′ are illustrated as being substantially orthogonal to the axis ofthe first flow containing the plurality of particles in otheralternative embodiments, the first inlet 135 and the second inlet 135′may intersect in the deformation region 140 in an off-axis manner yetnot be substantially orthogonal to the axis of first flow.

In one embodiment of FIG. 9A, the fluid that enters the first inlet 135and the second inlet 135′ are siphoned off from an upstream channel thatcontains the focused particles as seen in FIG. 9B. Because the particlesare aligned in the center of the channel due to focusing, side streamscan be siphoned off the main flow while letting the focused particlesremain in the central branch channel 122″. This particular embodiment isreferred to as hydropipette aspiration (HA). The branch channels 135,135′ are subsequently returned to apply a pinching flow at thedeformation region 140. Particles are then deformed by the rejoiningcell-free (in some embodiments) “sheath” fluid. In contrast withdeformability cytometry (DC) whereby cells are subject to a head-on flowand quickly slowed and then accelerated in a transverse direction, theHA device and method is able to achieve a much higher particlethroughput. For example, a throughput of 65,000 cells/sec. has beenachieved using this design compared to a throughput of around 2,000cells/sec. achieved using the DC design.

FIG. 9B illustrates an example of a fluidic pathway that utilizes theoff-axis configuration illustrated in FIG. 9A. As seen in FIG. 9B, fluidcontaining the plurality of particles passes first through a filter 190.The outlet of the filter 190 is coupled to a delivery region 130 asdescribed herein that is used to substantially focus the plurality ofparticles along a common axis as seen inset image at point c in thefluidic pathway. The particles then enter a trifurcation 125″. Theparticles continue along via central branch channel 122″ while a portionof the substantially particle-free fluid is shunted to inlets 135, 135′where they recombine with the central branch channel 122′ in thedeformation region 140 to squeeze and deform the particles asillustrated. The particles continue on in the same direction to outlet106.

Furthermore, the first opposing flow and the second opposing flow can begenerated from the sample volume by siphoning portions of the samplevolume (e.g., into a trifurcation or bifurcation that rejoins at thedeformation region), or by flows (e.g., injected sheath flows) notgenerated from the sample volume. In this example, the particles arethus compressed in a direction substantially orthogonal to a directionin which the particle flows, and extends along the direction in whichthe particle flows. In the configuration provided in this example,particles do not undergo substantial deceleration (e.g., slow down orstop) upon entering the deformation region 140, and the throughput ofthe system 100 can be increased because multiple particles of theplurality of particles can enter the deformation region 140simultaneously. Furthermore, a variable range of forces used to deformparticles of the plurality of particles can be generated by the firstopposing flow and the second opposing flow, by modulating flowparameters of any one or more of the first flow, the first opposingflow, and the second opposing flow. Small forces used to deform theparticles can, in particular, be interesting for probing intrinsicparticle properties and/or properties of smaller particles (e.g., <10micrometers in diameter), and can provide insight into membraneelasticity, particle relaxation behavior and other properties ofparticles that are difficult to assess with large deformation forces.

In still other variations, the delivery region 130 and the deformationregion 140 can be configured using any suitable number of branches andin any other suitable manner that enables focusing of the plurality ofparticles and deformation of the plurality of particles. For example, avariation of the fluidic pathway 120 can comprise multiple deliveryregions 130 configured upstream and downstream of a deformation region140, such that the plurality of particles is focused before and afterdeformation. In other examples, multiple branches (e.g., more than twobranches) can be configured to convene upon the deformation region 140,in order to provide alternative modes of deformation. In still otherexamples, the plurality of particles can be configured to enter a firstdeformation region 140 configured to provide deformation from flows thatare orthogonal to a direction of the flow carrying the plurality ofparticles, and can be configured to subsequently enter a seconddeformation region 140′ configured to provide a deformation force from aflow that is anti-parallel to a flow carrying the plurality ofparticles. Additionally or alternatively, the plurality of particles canbe configured to be actively sorted or directed (e.g., by focusing, byflow diversion, based upon channel resistance), into a specific outlet106. This example could facilitate additional processing of theplurality of particles, as enabled by uniform flow conditions within theadditional delivery region 130 and/or active sorting downstream of thedeformation region 140.

FIG. 9C illustrates the fluidic pathway of FIG. 9B with the resistanceslabeled for the central branch channel 122″ and inlets 135, 135′ for thedeformation region 140 according to one design. As seen in FIG. 9C,R2=1.7*R1. This leads to a decreased fraction of flow down the centralbranch channel 122″ but allows for sufficient Reynolds number forefficient inertial focusing. The outer branches have a lower resistanceto allow for a higher flow rate and velocity. This enables a largersqueezing flow on the cells as they pass through the deformation region140.

FIG. 9D illustrates an embodiment of a fluidic pathway that combinesoff-axis squeezing at a first deformation region 140 followed by asecondary deformation region 140′ in which particles are subject todeformation at an intersection of opposing flows. In this embodiment,fluid containing the plurality of particles passes first through afilter 190. The outlet of the filter 190 is coupled to a delivery region130 as described herein that is used to substantially focus theplurality of particles along a common axis. The particles then enter ajunction 125′″ of five (5) branch channels 141, 141′, 141″, 141′″,141″″. Central branch channel 141 contains substantially all theparticles. Outer branch channels 141′, 141″, 141′″, and 141″″ aresubstantially free of particles and contain portions of fluid shuntedfrom junction 125′ Inner branches 141′, 141′″ recombine with the centralbranch channel 141 in an off-axis manner to squeeze the particles at thefirst deformation region 140. The particles continue to a seconddeformation region 140′ whereby fluid from branch channels 141″, 141″″recombine and intersect with the central branch channel 141 in anopposing flow. Particles passing through this second deformation region140′ can then exit the fluidic pathway via one or both outlets 106′,106″.

FIG. 9E illustrates a simplified resistor diagram of the combined designof FIG. 9D that uses off-axis squeezing of particles (hydropipetteaspiration or “HA”) in conjunction with deformability cytometry (“DC”).Tuning of resistance is used to ensure equal flow through the twobranches of channels creating the extensional flow (RDC and RHA). Inthis embodiment, RDC≈RHA.

FIG. 9F illustrates another embodiment of a fluidic pathway in whichhydropipette aspiration is combined with rapid inertial solutionexchange for integrated sample preparation and analysis. In theembodiment of FIG. 9F, a solution containing a plurality of particles isdelivered to inlet 104 which then passes through a filter 190. Theoutlet of the filter 190 terminates in a bifurcation 142 that thenrecombine in an anti-parallel, off-axis junction J with a centralchannel 143 fluidically coupled to a wash inlet 104′. As seen in FIG.9F, a filter 190′ is interposed between the outlet of the wash inlet104′ and the central channel 143. The central channel 143 continuesuntil another trifurcation 144 that results in a first branch channel145, a second branch channel 146 and a continuation of the centralchannel 143 which may include a focusing or delivery region as describedherein. The first and second branch channels 145, 146 are configured tosiphon off a portion of fluid flow within the central channel 143. Inthe embodiment of FIG. 9F, the first and second branch channels 145, 146act as waste channels which are fluidically coupled to outlet 106. Adeformation region 140 is formed downstream of the trifurcation 144 byan intersection of the central channel 143 as well as first and secondside channels 147, 148. The first and second side channels 147, 148 areoriented substantially orthogonal to the central channel 143 and arecoupled to an inlet 159 that is configured to be fluidically coupled toa pressurized source of fluid. In this regard, sheathing fluid entersinlet 159 and passes into channels 147, 148 which then recombine withthe central channel 143 at the deformation region 140. This fluid floweffectuates side squeezing or sheathing of the particles as describedherein. After passing through the deformation region 140, the particlescan then exit the device via outlet 106′.

FIG. 9G illustrates series of magnified images of selected regions ofthe device of FIG. 9F. As seen in FIGS. 9F and 9G, in this particularexample, a solution containing a mixture of cells (e.g., a blood samplecontaining a mixture of cells) is delivered to the inlet 104. A washsolution is delivered to the wash inlet 104′. The wash solution mayinclude, for example, phosphate buffered saline (PBS). At the junctionJ, the outer channels that combine with the central channel 143. Afterthe junction J, in the central channel 143 size-dependent lift forcesact upon the larger cells (e.g., cancer cells) to transfer them to thecentral wash solution contained in the central channel 143. Stillreferring to FIG. 9F, when the cells reach the trifurcation 144, thesmaller blood cells (e.g., white blood cells) are siphoned off to thefirst and second branch channels 145, 146. The cancer cells continue onin the central channel 143 past the trifurcation 144. Meanwhile, duringoperation of the device, a solution such as PBS is delivered to theinlet 159 using a pump or the like to create the squeezing sheathingflow at the deformation region 140. At or adjacent to the deformationregion 140, the cells can be imaged using a detection module 150(described in more detail below) that can generate a morphology datasetand/or fluorescent dataset for the cells.

1.3 System—Detection Module

As shown in FIG. 1, the detection module 150 includes an imagingsubsystem 151 and a fluorescence subsystem 156, and functions togenerate a morphology dataset characterizing deformation of eachparticle, and a fluorescence dataset characterizing fluorescence of eachparticle in the plurality of particles. Preferably, the deformationregion 140 substantially coincides with a field of view of the at leastone of the imaging subsystem 151 and the fluorescence subsystem 156, andadditionally, the detection module 150 is preferably configured tocapture a field of view extending beyond the deformation region 140. Assuch, the imaging module 151 and the fluorescence module 155 can beconfigured to focus upon any suitable region including and extendingbefore or beyond the deformation region 140. For example, in someembodiments, fluorescent images are obtained prior to the particlesentering the deformation region 140. Preferably, the detection module150 generates the morphology dataset and the fluorescence datasetsimultaneously; however, the detection module 150 can alternatively beconfigured to generate the morphology dataset and the fluorescencedataset non-simultaneously (e.g., sequentially). In variations whereinthe detection module 150 generates the morphology dataset and thefluorescence dataset simultaneously, the detection module 150 ispreferably configured such that light (e.g., white light) used togenerate the morphology dataset does not interfere with generation ofthe fluorescence dataset. Interference can take the form of unwantedexcitation of fluorescent labels and/or saturation of fluorescencedetectors (e.g., photodetectors) during generation of the fluorescencedataset.

The imaging subsystem 151 functions to generate a morphology datasetcharacterizing deformation of the particles. Referring now to FIG. 10A,the imaging subsystem 151 preferably comprises a first light source 152and a first filter 153 configured to transmit light from the first lightsource 152, through the deformation region 140 and onto an objectivelens 154, the objective lens configured to magnify light from thedeformation region onto an image sensor 155 for generating themorphology dataset. The imaging subsystem 151 can additionally compriseany suitable number of lenses, for example, for focusing light from thefirst light source 152 through the first filter 153, for focusing lightfrom the first filter 153 onto the deformation region 140, and forfocusing light from the objective lens 154 onto the image sensor 154.The lenses thus function as collection and condensing optics elements,and preferably comprise aspheric lenses; however, the lenses canalternatively comprise plano-convex lenses and/or any other suitablelenses configured to collect and condense light.

The first light source 152 functions to provide enough illumination forgenerating a morphology dataset at the image sensor 155, withoutproducing unwanted excitation of fluorescent labels at the plurality ofparticles and/or saturation of a fluorescence detector (e.g.,photodetector). As such, the first light source 152 preferably providesa specified range of wavelengths that minimally overlaps with range ofwavelengths of fluorescent emission generated in response tofluorescence subsystem 156. The first light source 152 thus preferablyprovides a sufficient intensity of light that enables properillumination during short exposure times used in high-speed image datacapture. As such, the first light source 152 can be filtered by thefirst filter 153, in order to reduce interference at a photodetector ofthe fluorescence subsystem 156 while still providing sufficientillumination at the image sensor 155. In a first variation, the firstlight source 152 is a xenon light source, which can be used inhigh-speed imaging applications and fluorescence imaging. Alternatively,the first light source 152 can comprise a halogen light source and/orany other suitable light source in other variations. Furthermore,variations of the detection module 150 can includeinterchangeable/adjustable light sources, in order to provide varyingranges of light wavelengths, varying intensities of light, and/or anyother suitable varying light parameter.

The first filter 153 functions to filter light from the first lightsource 152 and to transmit filtered light toward the deformation region140, in order to avoid spectral overlap between the imaging subsystem151 and the fluorescence subsystem 156. As such, the first filter 153 ispreferably coaxially aligned with the first light source 152, in orderto properly filter light from the first light source 152. Preferably,the first filter 153 is a bandpass filter configured to only pass lightthat does not excite fluorophores at the plurality of particles, andadditionally, to only pass light that is not detected by a photodetectorof the fluorescence subsystem 156. In a specific example, the firstfilter is configured to filter out wavelengths around 532 nm and around580 nm, in order to not excite fluorescent labels bound to particles andto avoid light interference at a photodetector of the fluorescencesubsystem 156 respectively. In alternative variations the first filter153 can comprise a lowpass filter, a highpass filter, and/or any othersuitable filter for filtering interfering light wavelengths.Furthermore, variations of the detection module 150 can compriseinterchangeable filters for filtering light from the first light source152.

The objective lens 154 functions to receive light from the first filter153 and passing through the deformation region 140 and to magnify lightonto an image sensor 155, in order to facilitate generation of amorphology dataset characterizing deformation of each particle in theplurality of particles. The objective lens 154 is preferablysubstantially aligned between the first filter 153 and the image sensor;however, the objective lens 154 can alternatively have any othersuitable configuration relative to other elements of the detectionmodule 150. The objective lens is preferably characterized by amagnification that enables an entire deformed particle of the pluralityof particles to be captured within a window defined by the image sensor155, wherein the desired magnification depends upon the focal length ofthe objective lens and/or focal length(s) of any additional opticselement(s) (e.g., tube lens), and the position of the image sensor 155relative to the objective lens and/or optics element(s). In a specificexample, the objective lens provides a 10× magnification; however, inother variations, the objective lens can provide any other suitablealternative magnification. In variations, the detection module 150 caninclude interchangeable/adjustable objective lenses 154, in order toprovide an adjustable magnification. Different levels of magnificationcan enhance the morphology dataset generated at the image sensor 155, byproviding, for example, magnification of features not seen at allmagnification levels.

The image sensor 155 functions to receive light from the deformationregion 140 and passing through the objective lens 154, in order togenerate a morphology dataset characterizing deformation of eachparticle in the plurality of particles. Preferably, the image sensor 155is substantially aligned with the objective lens 154; however, the imagesensor 155 can have any other suitable configuration relative to otherelements of the detection module 150. The image sensor 155 can beintegrated into a high-speed/high frame-rate imaging module (e.g.,camera), configured to generate image data that captures multiple stagesof deformation for each particle in the plurality of particles. Anexample of a high-speed/high frame-rate imaging module is the digitalhigh-speed video camera, Phantom v7.3 (Vision Research, Inc., Wayne,N.J., USA) which has a frame rate between 6,688 fps to 500,000 fps. Assuch, specifications of the image sensor 155 and the light source arepreferably codependent in order to provide sufficient light parameters(e.g., intensity) for image data generation. The image sensor 155 cancomprise a variation of the image sensor described in U.S. Pub. No.2013/0177935, entitled “Method and Device for High Throughput CellDeformability Measurements”, which is incorporated herein in itsentirety by this reference; however, the image sensor 155 can compriseany other suitable image sensor for generating the morphology dataset.

The fluorescence subsystem 156 functions to generate a fluorescencedataset characterizing the fluorescence (or absence of fluorescence) ofeach particle in the plurality of particles. The fluorescence subsystem156 can thus comprise a second light source 157 and a second filter 158configured to transmit light from the second light source 157, through afiber optic unit 159, through a portion of the fluidic pathway 120, andonto an objective lens 154, the objective lens 154 configured to magnifylight from the fluidic pathway 120 onto a photodetector 160 forgenerating the fluorescence dataset. Light from the fluidic pathway 120can further be passed through a third filter 161 prior to reception atthe photodetector 160, in order to reduce or eliminate effects ofinterfering wavelengths of light.

The second light source 157 functions to provide excitation wavelengthsof light, and to transmit light at excitation wavelengths toward eachparticle in the plurality of particles in a portion of the fluidicpathway 120. The second light source preferably directs light toward thesecond filter 158 and the fiber optic unit 159, onto a portion of thefluidic pathway 120, such that fluorescent labels bound to particlespassing through the portion of the fluidic pathway 120 are excited byexcitation wavelengths of light. In response, the excited fluorescentlabels emit emission wavelengths of light, indicative of biomolecularcharacteristics of the particles, which can be detected at aphotodetector 160. The second light source 157 is preferably a lightsource that provides a specific excitation wavelength of light, and canbe a laser (e.g., a 532 nm laser). However, the second light source 157can alternatively be configured to provide a range of excitationwavelengths of light. In one variation, the second light source 157 canbe a broad-spectrum light source (e.g., white light LEDs) that transmitslight through at least one excitation filter to generate a specificwavelength or range of wavelengths of light for fluorescent labels(s)excitation. In variations including the excitation filter(s) and abroad-spectrum light source, the excitation filter(s) can beinterchangeable in order to provide an adjustable excitation wavelengthor an adjustable range of excitation wavelengths.

The second filter 158 functions to modify a parameter of lighttransmitted from the second light source 157, in order to conditionlight provided by the second light source 158. The second filter 158 ispreferably aligned between the second light source 157 and the fiberoptic unit 159; however, in variations omitting the fiber optic unit159, the second filter 158 can be aligned with the second light source157 or can have any other suitable configuration. The second filter 158is preferably a neutral density filter, which is configured to modify orreduce an intensity of light transmitted from the second light source157. As such, the neutral density filter can function to prevent signalsaturation due to high-intensity light, and can additionally function toprotect sensitive elements of the detection module 150 fromhigh-intensity light. The second filter 158 can, however, comprise anyother suitable filter for conditioning light from the second lightsource 157.

The fiber optic unit 159 functions to redirect light transmitted throughthe second filter 158 from the second light source 157, in order tosatisfy space requirements of the system 100. Furthermore, the fiberoptic unit 159 can function to alter a beam shape (e.g., by a fibercollimator to produce a more spatially uniform beam), and can facilitatetranslation by coupling to a mount for fine resolution translation inone or more directions (e.g., two dimensions by an x-y mount). As such,the fiber optic unit 159 can include a fiber coupler coupled to a fiberoptic-fiber probe assembly that allows light to be transmitted throughthe fiber-optic-fiber probe assembly. The fiber probe is preferablyconfigured to direct light into a portion of the fluidic pathway throughwhich the plurality of particles pass, such that fluorescent labelsbound to the plurality of particles can be properly excited. The portionof the fluidic pathway can comprise the deformation region 140, suchthat the detection module 150 is configured to simultaneously or nearlysimultaneously capture deformation and fluorescence characteristics atthe same location along the fluidic pathway; however, the portion of thefluidic pathway can alternatively comprise any other suitable region ofthe fluidic pathway, for example, a region upstream of the deformationregion 140 and downstream of a delivery region 130, or any othersuitable region of the fluidic pathway. In one variation, light from thesecond light source 157 can be directed toward a region immediatelyupstream of the deformation region (e.g., 100 micrometers to 1 mmupstream), wherein flow conditions are sufficiently uniform. In somevariations, wherein space is less of a constraint, the fluorescencemodule 156 can omit the fiber optic unit 159, light from the secondlight source 157 through the second filter 158 can be transmitteddirectly in a straight line from the second light source 157 to theportion of the fluidic pathway 120. Some variations of the fluorescencesubsystem 156 can, however, omit the fiber optic unit 159 and insteadcomprise beam steering mirrors to translate a beam provided by thesecond light source 157 in multiple dimensions and/or a movable stage(e.g., x-y stage) configured to facilitate translation of a beam in oneor more directions.

Similar to the objective lens of the imaging subsystem 151, theobjective lens 154 functions to receive light from the second filter 158passing through the portion of the fluidic pathway 120, and to magnifylight onto a photodetector 160, in order to facilitate generation of afluorescence dataset characterizing fluorescence of each particle in theplurality of particles. The objective lens 154 can be positioned betweenthe fiber probe of the fiber optic unit 159 and the photodetector 161 inany suitable configuration relative to other elements of the detectionmodule 150. The objective lens is preferably characterized by amagnification that enables an entire fluorescing particle of theplurality of particles to be captured within a window defined by thephotodetector 160, wherein the desired magnification depends upon thefocal length of the objective lens and the position of the photodetector160 relative to the objective lens 154. In a specific example, theobjective lens provides a 10× magnification; however, in othervariations, the objective lens can provide any other suitablealternative magnification. In variations, the detection module 150 caninclude interchangeable/adjustable objective lenses 154, in order toprovide an adjustable magnification.

The photodetector 160 functions to receive light emitted upon excitationof fluorescent labels bound to particles of the plurality of particles.The photodetector 160 additionally functions to facilitate generation ofa fluorescence dataset characterizing fluorescence characteristics foreach particle in the plurality of particles. As such, the photodetector160 is preferably configured to detect ultraviolet, visible, andinfrared light, emitted from excited fluorescent labels. In onevariation, the photodetector 160 can comprise a photomultiplierconfigured to operate by a photoelectric effect upon reception ofincident light; however, in other variations, the photodetector 160 caninclude any other suitable photodetector configured to detect anysuitable wavelength of light, by any other suitable mechanism.

As described earlier, the fluorescence module 150 can include a thirdfilter 161 configured to filter light prior to reception at thephotodetector 160. The third filter 161 thus functions to reduce oreliminate any effect of interfering light generated from any source(e.g., the first light source 152). Preferably, the third filter 161 issubstantially aligned with the photodetector 160, such that incidentlight on the photodetector 160 is configured to pass through the thirdfilter 161. Additionally or alternatively, the third filter 161 can beconfigured along any suitable portion of a light path from the objectivelens 154 to the photodetector 160. The third filter 161 preferablycomprises a bandpass filter; however, the third filter 161 canalternatively or additionally comprise a lowpass filter or a highpassfilter.

Preferably, the imaging subsystem 151 and the fluorescence subsystem 155are integrated, in order to reduce space and cost demands of thedetection module 150. As such, in some variations, the imaging subsystem151 and the fluorescence subsystem 155 can share elements. In one suchvariation, the imaging subsystem 151 and the fluorescence subsystem 155can share a single light source, with flow parameters correspondinglyadjusted to ensure that there is only a single particle at a time in anillumination spot provided by the light source. In other variations,other elements can be additionally or alternatively be shared betweenthe subsystems 151, 155.

In an example, as shown in FIG. 10A, the imaging subsystem 151 and thefluorescence subsystem 155 share an objective lens 154 thatsimultaneously receives and transmits light originating from the firstlight source 152 and the second light source 157 toward an image sensor156 and a photodetector 160, respectively. In the example, the imagingsubsystem 151 and the fluorescence subsystem 155 further share adichroic mirror 162 configured to transmit specific wavelengths of lightfrom the objective lens 154, and to reflect other wavelengths of lightfrom the objective lens 154 (e.g., to another light sensing module). Thedichroic mirror 162 can be configured to reflect light emitted byfluorescent labels, in response to excitation, toward the photodetector160 to generate the fluorescence dataset, and to transmit light from thefirst light source 152 directly toward the image sensor 156 to generatethe morphology dataset. Alternatively, the dichroic mirror 162 can beconfigured to transmit light emitted by fluorescent labels toward thephotodetector 160 and to reflect light from the first light sourcetoward the image sensor 156. The dichroic mirror 162 is preferably ashort-pass dichroic mirror, but can alternatively be a long-passdichroic mirror or any other suitable dichroic mirror.

In another example, as shown in FIG. 10B, the imaging subsystem 151 andthe fluorescence subsystem 155 share an objective lens 154 thatsimultaneously receives and transmits light originating from the firstlight source 152 and the second light source 157 toward an image sensor156 and a photodetector 160, respectively. In this example, thedetection module 150 includes a first xenon light source 152 configuredto transmit light through a lowpass filter 153 and a plurality of lensesseparated by an aperture and a lowpass filter 153, toward thedeformation region 140 of the fluidic pathway 120, and through a 10×objective lens to be reflected off of a first and a second dichroicmirror 162, 162′ toward an image sensor 156. In this example, thedetection module 150 further includes a second 532 nm laser light source157 configured to transmit light through the first dichroic mirror 162,to be reflected off of the second dichroic mirror 162′ toward the 10×objective lens. Excitation light from the second light source 157 isconfigured to focus upon the deformation region 140, and light emittedfrom fluorescent labels at the deformation region is configured to betransmitted back through the second dichroic mirror 162′, through abandpass filter and a lens and a bandpass filter, toward a photodetector160. In variations of this example, light emitted from fluorescentlabels at the deformation region 140 can be transmitted through thesecond dichroic mirror 162′ and toward additional photodetectors 160′,160″ by additional dichroic mirrors 162′″, 162″″, bandpass filters, andlenses, wherein the photodetectors 160, 160′, and 160″ are configured toreceive different wavelengths (or ranges of wavelengths) of light.

In still another example, as shown in FIG. 100, the imaging subsystem151 and the fluorescence subsystem 155 share an objective lens 154 thatsimultaneously receives and transmits light originating from the firstlight source 152 and the second light source 157 toward an image sensor156 and a photodetector 160, respectively. In this example, thedetection module 150 includes a first light source 152 configured totransmit light through a lowpass filter 153 and a plurality of lensesseparated by an aperture and a lowpass filter 153, toward thedeformation region 140 of the fluidic pathway 120, and through a 10×objective lens 154 and a first short pass dichroic mirror 162 toward animage sensor 156. In this example, the detection module 150 furtherincludes a second 532 nm laser light source 157 coupled to a fiber probeand configured to transmit light through collimating optics, through abeam steering element (e.g., a set of mirrors, as in FIG. 10C, or an x-ytranslating fiber mount), through a second short pass dichroic mirror162, to be reflected off of the first short pass dichroic mirror andthrough the objective lens 154 to the deformation region 140. Lightemitted from fluorescent labels at the deformation region is thenconfigured to pass into the objective lens 154, to be reflected off thefirst short pass dichroic mirror 162 and the second dichroic mirror 162to a set of long pass dichroic mirrors 162′. The set of long passdichroic mirrors is configured to reflect and transmit specificwavelengths of light, through band-pass filters 161, toward specificphotodetectors 160 for fluorescence detection, as shown in FIG. 10C.

In other variations, the detection module 150 can include any othersuitable element(s) and/or configuration of elements that allowssimultaneous or near simultaneous generation of the morphology datasetand the fluorescence dataset. In examples, the detection module cancomprise any one or more of a beam splitter, an aperture, an additionaldichroic mirror, a collimator, any number of lenses, and any othersuitable element configured to manipulate light from a light source.Furthermore, any element can be coupled to an actuator (e.g., manual,automatic actuator) that enables alignment of optics and/or adjustmentof focal lengths. In one example, the objective lens 154 can be coupledto a linear actuator (e.g., a z-axis control) that enables adjustmentalong one or more axes. Additionally or alternatively, the substrateitself 110 can be coupled to an actuator (e.g., by a stage) thatprovides linear actuation along one or more axes (e.g., by an x-ycontrol).

1.3.1 System—Detection Module Alternatives

In some embodiments, the detection module 150 can additionally oralternatively include a one-dimensional detection module 163 configuredto facilitate an increase in data acquisition rates and a decrease inanalysis times. The one-dimensional detection module 163 functions toenable extraction of particle deformation characteristics, withoutgeneration of two-dimensional or three-dimensional data, in order togenerate a morphology dataset.

In a first variation, as shown in FIG. 11A, the one-dimensionaldetection module 163 comprises an optical mask including a set of slits164 configured to facilitate generation of particle transit timemeasurements with an increased signal-to-noise ratio. In the firstvariation, the optical mask preferably includes at least one slitsituated upstream of the deformation region 140 and at least one slitsituated downstream of the deformation region, which enables detectionof a difference in a particle dimension (e.g., cell length) before andafter particle deformation. A photodetector 169 configured to receivelight through the optical mask, and to generate an electrical signal(e.g., a voltage drop) upon a change in incident light produced by aparticle passing a slit of the optical mask, can be used to provide acorrelation between an electrical signal (e.g., voltage drop, durationof a voltage drop) and a particle dimension (e.g., cell length). In thefirst variation, the slit width is governed by an anticipated particledimension, and in specific examples, is preferably smaller than thesmallest expected cell size in order to directly infer a cell dimensionfrom the one-dimensional detection module 163. However, the optical maskcan alternatively include a slit with a width greater than ananticipated particle dimension (e.g., to facilitate optical maskfabrication), and signals generated by a photodetector cooperating withthe optical mask can be configured to produce deformation measurementsbased upon deconvolution with mean transit signal characteristics. In analternative to the first variation, the one-dimensional detection module163 comprises an optical mask including a set of patterns and aphotodetector 169 configured to receive light through the optical mask,and to generate an electrical signal (e.g., a voltage drop, duration ofa voltage drop) upon a change in incident light produced by a particlepassing a pattern of the optical mask. In this alternative, a signalproduced by the photodetector can be matched to a library of generatedsignals in order to extract particle dimensional parameters (e.g., celllength). However, the optical mask can alternatively include any otherfeatures configured to enable detection of a particle dimension withoutgeneration of two-dimensional or three-dimensional data.

In a second variation, the one-dimensional detection module 163 cancomprise a set of lenses 166 with a liquid waveguide 167 coupled to alight source 168 (e.g., fiber optic coupled to a light source) and adetector 169, wherein a light ribbon generated by light passing from thelight source 168, through the liquid waveguide 167, and through the setof lenses 166, can be used to generate transit time measurementsresulting from a voltage drop induced by a particle passing the lightribbon. The liquid waveguide 167 and the set of lenses 166 arepreferably integrated (e.g., physically coextensive) with the substrate110, as shown in FIG. 11B; however, the liquid waveguide 167 and/or theset of lenses 166 can alternatively be configured in any other suitablealternative manner. Furthermore, the light ribbon can be directeddirectly to the detector 169, such that the light source 168 is directlyopposed to the detector 169 as in FIG. 11B, or can be directed betweenthe light source 168 and the detector 169 in any other suitable manner(e.g., using positionally offset waveguides). In a specific example ofthe second variation, the liquid waveguide comprises high-refractiveindex oil (e.g., index=1.6), and the lenses have a refractive index of 1to facilitate generation of the light ribbon.

In a third variation, the one-dimensional detection module 163 cancomprise a detector 169 configured to enable generation of a particledimension measurement during deformation using forward and/orside-scatter measurements, as shown in FIG. 11C. Scattered-lightfeatures (e.g., profiles, parameters) detected as a particle enters andleaves the deformation region 140 can be used to infer particledeformation characteristics. For example, light scattering, as detectedby the detector 169, can produce a voltage drop that increases inmagnitude with increasing deformation, as shown in FIG. 11C. At least aportion of the third variation of the one-dimensional detection module163 can be integrated into the substrate 160; however, the thirdvariation of the one-dimensional detection module 163 can alternativelybe physically distinct from the substrate 110.

In still other embodiments, the detection module 150 can additionally oralternatively include a two-dimensional detection module 170 configuredto facilitate an increase in data acquisition rates and a decrease inanalysis times. The two-dimensional detection module 170 functions toenable rapid extraction of particle deformation characteristics basedupon alternative element compositions and/or configurations, in order togenerate a morphology dataset. An exemplary two-dimensional detectionmodule 170 is illustrated in FIG. 12A.

In one embodiment, the two-dimensional detection module 170 includes afirst position-sensitive detector 171 (PSD) configured to detect aparticle deformation along a first axis (e.g., x-axis deformation of aparticle) as the particle is deformed within the deformation region 140,and a second PSD 172, oriented orthogonally to the first PSD 171 andconfigured to detect a particle deformation along a second axis (e.g.,y-axis deformation of a particle) as the particle is deformed within thedeformation region 140. The first and the second PSDs 171, 172 are eachpreferably configured to generate an electrical signal (e.g., voltagedrop, duration of a voltage drop) indicative of a particle dimension(e.g., length) during particle deformation within the deformation region140, as shown in FIG. 12A. Signals provided by the first PSD can bepassed in a first channel and signals provided by the second PSD can bepassed in a second channel, and in an alternative variation, signalsprovided by the first PSD and the second PSD 171, 172 can be multiplexedin a single channel to reduce resource requirements during signalprocessing. In a specific example of the first variation, the first andthe second PSDs are defined by a 100 kHz bandwidth and a 2 micrometerspatial resolution, configured to enable deformation measurements withina 70 micrometer×70 micrometer region of the deformation region 140.

In a second embodiment, as shown in FIG. 12C, the two-dimensionaldetection module 170 comprises an image sensor 173 and a fieldprogrammable gate array (FPGA) 174 configured to cooperate with theimage sensor 173 to identify a particle event, and to selectivelytrigger signal capture of a particle undergoing deformation uponidentification of the particle event. The image sensor 173 is preferablyconfigured to capture image data at the deformation region 140, but canbe configured in any other suitable manner. In the second variation ofthe two-dimensional detection module 170, the detection module 150 canthus avoid collecting a substantial number of blank frames (i.e., framesnot providing any particle-related data), which significantly reducescomputational workload. In a specific application, the FPGA can beconfigured to screen a trigger region 175 upstream of the deformationregion 140, and upon identification of a particle within the triggerregion 175 (i.e., the particle event) by the FPGA, the image sensor 173can be configured to capture image data of the particle undergoingdeformation within a limited time window (e.g., 30 frames).

In a third embodiment, the two-dimensional detection module 170comprises an image sensor 176 configured to capture deformation of aparticle within the deformation region 140, a light source 177configured to emit light toward particles entering the deformationregion 140 at a location upstream of the deformation region, aphotodetector 178 configured to receive light from the light source 177,thus facilitating identification of a particle about to enter thedeformation region 140, and a strobe 179 configured to flash multipletimes in synchronization with motion of the particle within thedeformation region 140. Flashing of the strobe 179 thus enablescapturing of multiple positions and/or deformations of a particle withina single image frame, which allows a single image frame to provide moreuseful data related to particle deformation characteristics. The strobecan be configured to flash multiple times, with a fixed time intervalbetween strobe flashes, and can alternatively be configured to flashwithout a fixed time interval between strobe flashes, as guided by thephotodetector 178. In a specific example of the third variation, theimage sensor 176 is characterized by a frame rate of 2,000-10,000 framesper second and a field of view of 150 micrometer×150 micrometer. Thelight source 177 in the example is a laser focused upstream of thedeformation region 140, and the photodetector 178 comprises at least oneof a photomultiplier tube (PMT) and an amplified photodiode configuredto detect scattered laser light produced when a particles passes throughthe laser beam. The scattered light, as detected by the photodetector178 in the specific example, is used to trigger the strobe 179 to flashtwice (e.g., with a 500 ns exposure time) with a fixed time intervalcorresponding to a time required for the particle (i.e., the particlescattering light from the laser) to transit between two positions aboutthe deformation region 140. In the specific example, each image framethus comprises information related to two positions and twomorphological characterizations of a particle undergoing deformation inthe deformation region 140.

Other alternative variations of the detection module 150 can include anyother suitable element(s) or combination of elements that enablemeasurement and detection of particle morphological data that yielddeformation based upon single-dimension acquisition and/ormulti-dimension acquisition.

1.4 System—Other Elements

Referring back to FIG. 1, the processor 180 functions to transform themorphology dataset into a set of deformation characteristicscharacterizing deformation of each particle in the plurality ofparticles, to transform the fluorescence dataset into a set offluorescence parameters characterizing biomolecular properties of eachparticle in the plurality of particles, and to generate an analysisbased upon the set of deformation characteristics and the set offluorescence parameters. Preferably, the morphology dataset and thefluorescence dataset are temporally synchronized, to facilitate matchingof image and fluorescence data with specific particles in the pluralityof particles; however, the image and the fluorescence datasets can besynchronized by any other metric. As such, the processor 180 preferablyincludes a first module 181 configured to extract a set of deformationcharacteristics from the morphology dataset, a second module 182configured to extract a set of fluorescence parameters from thefluorescence dataset, a third module 183 configured to synchronize themorphology dataset and the fluorescence dataset, and a fourth module 184configured to generate an analysis based upon the set of deformationcharacteristics and the set of fluorescence parameters. It should beunderstood that, in some alternative embodiments, any of the modules181, 182, 183, 184 may be combined with one another. The modules 181,182, 183, 184 can include instructions or algorithms executed by theprocessor 180. These modules 181, 182, 183, 184 may be stored in memoryor other data storage device operatively coupled to the processor 180.Further, while reference is made to a single processor 180 it should beunderstood that one or more additional processors 180 may functiontogether as a single processing unit.

The first module 181 functions to extract a set of deformationcharacteristics from the morphology dataset that can be used tosynchronize the morphology dataset with the fluorescence dataset, andcan be used to generate an analysis by the fourth module 184. The firstmodule 181 can extract the set of deformation characteristicscontinuously or near-continuously and in real time (e.g., such thatdeformation of a particle is tracked in real time); however, the firstmodule 181 can alternatively be configured to extract characteristicsnon-continuously and/or in non-real time. The set of deformationcharacteristics preferably provide morphological and/or structuralcharacteristics indicative of phenotype, such as nuclear size, chromatindecondensation, cytoskeletal disassembly/fluidization, and membranecompromise/lysis. The set of deformation characteristics can thusprovide information related to the cell membrane and/or the cellnucleus. In some variations, the set of deformation characteristics caninclude any one or more of: particle deformability (e.g., a ratio ofparticle length to width), particle elastic modulus (e.g., a ratio ofstrain measured in an initial high frequency deformation regime, tostress provided by a library of simulated fluid-induced stresses andparticle dimensions), particle viscosity (e.g., a measurement of strainrate in a low frequency deformation regime), particle hydrodynamicviscosity (e.g., based upon an inertial equilibrium position of aparticle), particle circularity (e.g., based upon a ratio of particleprojected area to particle projected perimeter), particle roughness(e.g., a standard deviation of particle radius measurements), particlesize (e.g., volume, area, diameter, etc.), particle topologicalcharacteristics, particle asymmetry, and any other suitablemorphological or structural characteristic, as shown in FIGS. 13A-13C.The first module 181 can also be configured to extract baselinemorphological particle characteristics, including one or more of:initial particle volume, initial particle diameter, initial particleasymmetry, and any other suitable baseline characteristic. Extractingparticle characteristics can be performed as in U.S. Pub. No.2013/0177935, entitled “Method and Device for High Throughput CellDeformability Measurements”, or in any other suitable manner. Invariations wherein the first module 181 is configured to extractbaseline morphological particle characteristics, the baselinecharacteristics can be used to normalize the set of deformationcharacteristics for each particle in the plurality of particles, and/orcan be used by the fourth module 184 to generate the analysis in anyother suitable manner. In one embodiment, the first module 181 outputs asequence indicator (e.g., frame number(s) of an image used to extract adeformation characteristic, time stamp, etc.) along with at least oneextracted deformation characteristic for each particle in the pluralityof particles; however, the first module can provide any other suitableoutput. In a specific example, the first module 181 is configured tooutput particle deformability along with the frame number(s) of an imageused to extract deformability.

The second module 182 functions to extract a set of fluorescenceparameters from the fluorescence dataset that can be used to synchronizethe fluorescence dataset with the morphology dataset, and can be used togenerate an analysis by the fourth module 184. The second module 182 canextract the set of fluorescence parameters continuously ornear-continuously and in real time (e.g., such that fluorescence of aparticle is tracked in real time); however, the first module 181 canalternatively be configured to extract characteristics non-continuouslyand/or in non-real time. The set of fluorescence parameters preferablyprovide characteristics indicative of biomolecular phenotype (e.g.,surface markers, nucleic acid composition, membrane integrity, receptorcharacteristics) and can include any one or more of: an intensity ofemitted light (e.g., average intensity, peak intensity), a wavelength ofemitted light, kinetic parameters of fluorescence, and any othersuitable fluorescence parameter. The second module 182 can also beconfigured to extract baseline fluorescence parameters (i.e., prior toparticle deformation), including one or more of: initial intensity(e.g., initial average or peak intensity), initial emitted wavelengthprior to deformation, initial kinetic parameter(s) prior to deformation,and any other suitable baseline parameter. In variations wherein thesecond module 182 is configured to extract baseline fluorescenceparameters, the baseline parameters can be used to normalize the set offluorescence parameters for each particle in the plurality of particles,and/or can be used by the fourth module 184 to generate the analysis inany other suitable manner. Preferably, the second module 182 outputs asequence indicator (e.g., time stamp, frame number(s) of an image usedto extract a fluorescence parameter, etc.) along with at least oneextracted fluorescence parameter for each particle in the plurality ofparticles; however, the first module can provide any other suitableoutput. In a specific example, the second module is configured to outputa continuous signal of intensity and time. For example, the signal maycomprise a continuous voltage signal from a PMT as described herein.Peaks corresponding to the detected fluorescent particles may beextracted from the generated dataset using the second module 182.

The third module 183 functions to synchronize the morphology dataset andthe fluorescence dataset. Preferably, the morphology dataset and thefluorescence dataset are output from the image sensor 155 and thephotodetector 160 using the same clock, such that time points across theimage data and the fluorescence data are substantially synchronized.Synchronization may be accomplished by subtracting an elapsed time thatcorresponds to the time delay when the particle passes from thefluorescence interrogation region to the morphology detection region. Insome variations, however, the morphology dataset and the fluorescencedataset may not be associated with the same clock, motivatingsynchronization of the morphology dataset and the fluorescence dataset.The third module 183 can be configured to perform any suitable signalconditioning step (e.g., noise removal by filtering and peak-finding).In one specific example, wherein the first module 181 is configured tooutput particle deformability along with a frame number of an image usedto extract deformability and the second module 182 is configured tooutput a continuous signal of intensity and time, the third module 183is configured to apply signal filters to remove signal noise and apply apeak-finding algorithm to identify a time-dependent sequence ofparticles. A sequence matching or cross-correlation algorithm, anexample of which is shown in FIG. 14, is then used to align the eventvs. time signals of the morphology dataset with the event vs. timesignals of the fluorescence dataset. In variations of the example,calibration particles (e.g., rigid or deformable fluorescent calibrationmicrospheres) characterized by identifiable deformability andfluorescence signatures can be used to synchronize the morphologydataset with the fluorescence dataset, irrespective of time stamps. Instill other variations, relationships between deformability (or anyother suitable deformation characteristic) and emitted fluorescenceintensity can be used to synchronize the sets of data. However, themorphology dataset and the fluorescence dataset can be synchronized inany other suitable manner.

The fourth module 184 functions to generate an analysis based upon theset of deformation characteristics and the set of fluorescenceparameters. The analysis can comprise a correlation between mechanicaland biochemical/biomolecular markers for the particles of interest,which can be used to identify mechanical (e.g., deformation)characteristics, fluorescence parameters, and/or combinations ofmechanical and fluorescence parameters useful for characterizingparticles of the plurality of particles. In specific applications, theanalysis generated by the fourth module 184 can be used to identifyactivation states of specific cell types (e.g., blood mononuclear cellactivation by mitogens or inflammatory processes, granulocyte activationwith cytokines or blood stream infections, as identified bydeformability and surface expression of activation markers), withimportant implications in label-free monitoring of diseases, diagnosisof diseases, treatment of diseases, and prediction of transplantrejection. In additional applications, the analysis generated by thefourth module 184 can be used to identify phenotypic connections betweenstem cells and cancers (e.g., Jurkat and HL60), used to identifydifferentiation indicators for stem cells, and used for identificationof subpopulations of cells within diverse populations of cells in bodyfluid samples from healthy or diseased patients (e.g., resting oractivated leuokocytes, PBMCs, and granulocytes as in blood, or pleuralfluid). As such, the fourth module 184 can be used to aggregate alibrary of data of multiple types of phenotypic markers (e.g.,mechanical, deformation, fluorescence, etc.) for a variety of biologicalparticles, using a high-throughput approach.

The fourth module 184 can be configured to conduct a statisticalanalysis (e.g., correlation, t-test, ANOVA, etc.), which functions toinvestigate relationships between deformation and fluorescenceparameters. Additionally or alternatively, classification and regressiontrees (CARTs) generated by the fourth module 184 can be used, withdeformation and fluorescence parameters used to enhance identification.Receiver operating characteristic (ROC) curves can be used to assess anability to correctly identify particles for purposes of generatingpredictive models. Furthermore, linear discriminate analyses (or othermachine learning approaches) can be used to identify similarities and/ordifferences between different sample volumes, which can be used, forexample, to stratify samples from different patients. In somevariations, the processor 180 can further be configured to render theanalysis at a user interface (e.g., as a flow cytometry 2D or 3D densityplot of single cells, etc.) such as a display or monitor.

As shown in FIG. 1, the system 100 can further comprise a filter 190located upstream of the delivery region 130. The filter 190 functions toseparate particles of interest from other particles or debris in thesample volume and to allow the particles of interest to pass into thefluidic pathway 120. Preferably, the filter 190 is configured between aninlet 104 and the fluidic pathway 102, such that the sample volume issubstantially filtered prior to delivery into the fluidic pathway 120,delivery region 130, and/or deformation region 140. Additionally oralternatively, the system 100 can include a filter 190 positioned at anyother suitable location of the system 100, and/or any suitable number offilters in any other suitable configuration. The filter 190 preferablyseparates the particles based upon size (e.g., using suitably sizedpores in a porous structure or mesh); however, the filter 190 canalternatively separate particles of interest from other particles in thesample volume based upon any other suitable separation mechanism (e.g.,chemical, affinity moiety, electric, magnetic, etc.).

Also shown in FIG. 1, the system 100 can further comprise a processedsample volume receiver 195, which functions to receive a processedsample fluid from an outlet 106 of the substrate 110. As brieflydescribed earlier, the processed sample volume receiver 195 can be awaste chamber configured to fluidly couple to the outlet 106 to collectthe processed sample fluid as waste. Furthermore, the waste chamber canbe integrated (e.g., physically coextensive, of unitary construction)with the substrate 110 in any suitable manner. Alternatively, theprocessed sample volume receiver 195 can be configured to collect andtransmit the processed sample volume, including the plurality ofparticles, to another module for additional assays and analyses. Assuch, the processed sample volume receiver can comprise one or moreconduits and/or valves that facilitate sample transmission. In stillother variations, the processed sample volume receiver 195 can be acomposite receiver that receives a portion of the sample volume aswaste, and facilitates collection of another portion of the samplevolume for further analyses.

In some variations, the system 100 can further comprise a storage module197 with accessible memory, which functions to receive and/or store atleast one of the morphology dataset, the fluorescence dataset, ananalysis, system 100 parameters (e.g., flow parameters, detection moduleparameters, etc.), sample volume identifiers (e.g., name, contents,date), and module algorithms. The accessible memory permits a user toaccess stored information about sample runs using the system 100 and thesystem parameters that were utilized during those runs. Any storedinformation is preferably accessible by a user and/or any other suitableentity. The storage module can be implemented using any suitablecomputing device (e.g., desktop computer, hardware storage device,server, cloud).

The system 100 can, however, include any other suitable element(s) orcombination of elements that facilitate the deformation, assaying,and/or analysis of particles of a sample volume. As a person skilled inthe art will recognize from the previous detailed description and fromthe figures and claims, modifications and changes can be made to theembodiments of the system 100 without departing from the scope of thesystem 100.

2. Method

As shown in FIG. 15, a method 200 for deforming and analyzing aplurality of particles carried in a sample volume includes: receivingthe sample volume including the plurality of particles S210; diverting afirst portion of the sample volume in a first flow and a second portionof the sample volume in a second flow, opposed to the first flow,wherein an intersection of the first and the second flows defines adeformation region S220; focusing the plurality of particles into thedeformation region S230; generating a morphology dataset characterizingdeformation of each particle in the plurality of particles within thedeformation region S240; generating a fluorescence datasetcharacterizing fluorescence of each particle of the plurality ofparticles within the deformation region S250; and outputting an analysisof the plurality of particles based at least in part on the morphologydataset and the fluorescent dataset for the plurality of particles S260.

The method 200 functions to enable the deformation of single particlesin a high-throughput and consistent manner, with the ability tosimultaneously generate and analyze multiple data types characterizingthe single particles. Preferably, the method 200 further functions toenable the generation of data that directly correlates surfacebiomarkers of phenotype with mechanical properties at thesingle-particle level. This can allow the generation of a directquantitative comparison between biomolecular properties and mechanicalproperties. Preferably, the method 200 is used to process and analyzebiological particles, such as cells, and in specific applications, themethod 200 can be used to analyze leukocyte activation, stem celldifferentiation, and cancer cell malignancy by way of correlatingcellular deformation with biomolecular phenotypes using fluorescenceassays. However, the system 100 can alternatively be used to process,deform, and analyze any other suitable biological particle ornon-biological particle using any other suitable analysis.

Block S210 recites: receiving the sample volume including the pluralityof particles, and functions to receive a sample volume, including theplurality of particles, to initiate processing and analysis of theplurality of particles. The sample volume is preferably received at aninlet of a substrate, using a pump, as in an embodiment of the system100 described above; however, the sample volume can be received and/ordelivered in any other suitable manner. In some variations, Block S210can further include filtering the sample volume S212, as shown in FIG.15, which functions to separate particles of interest from otherparticles in the sample volume and to allow the particles of interest topass into a fluidic pathway for further processing and analysis. BlockS212 can be implemented using any suitable variation of the filterdescribed above, or using any other suitable method of separatingparticles of interest from other particles in a sample volume.

Block S220 recites: diverting a first portion of the sample volume in afirst flow and a second portion of the sample volume in a second flow,opposed to the first flow, wherein an intersection of the first and thesecond flows defines a deformation region. Block S220 functions togenerate opposing flows configured to deform each particle in theplurality of particles. Block S220 is preferably implemented at anembodiment of the fluidic pathway of the system 100 described above,wherein the fluidic pathway includes at least two branches configured togenerate the first and the second flows from the sample volume.Additionally or alternatively, an injected flow, not derived samplevolume, can be used to generate at least one flow in the opposing flows.However, Block S220 can be implemented using any other suitable methodof generating opposing flows, at least partially from a sample volume.In some variations, Block S220 can include diverting a first portion ofthe sample volume in the first flow, wherein the first flow comprisessubstantially all of the particles of interest and diverting a secondportion of the sample volume in the second flow, wherein the second flowis substantially free of particles of interest; however, in othervariations, the first flow and the second flow can both comprise asubset of the plurality of particles.

Block S230 recites: focusing the plurality of particles into thedeformation region, and functions to transmit the plurality ofparticles, along at least one streamline into the deformation region,such that each particle in the plurality of particles experiencesuniform flow conditions prior to deformation within the deformationregion. Block S230 is preferably implemented at a delivery region in anembodiment of the system 100 described above, but can be implemented atany other suitable portion of a fluid pathway configured to focusparticles. Preferably, any flow including particles of the plurality ofparticles is focused into the deformation region in Block S230; however,in alternative variations, Block S230 can omit focusing of any subset ofthe plurality of particles, and/or focusing of flows not includingparticles of the plurality of particles. In one embodiment, focusing inBlock S230 includes focusing using inertial focusing in at least one ofa confined curved channel and a channel including a set of heightrestrictions, as described above; however, focusing in Block S230 cancomprise any one or more of: hydrodynamic focusing, focusing using asheath fluid, dielectrophoretic focusing, ultrasonic focusing, magneticfocusing, and any other suitable focusing method.

Block S240 recites: generating a morphology dataset characterizingdeformation of each particle in the plurality of particles within thedeformation region, and functions to generate a dataset that can be usedto extract a set of deformation characteristics for generation of ananalysis based upon deformation characteristics. The morphology datasetis preferably generated in Block S240 using an embodiment of thedetection module and imaging subsystem described above; however, themorphology dataset can additionally or alternatively be generated usingany suitable module including an image sensor configured to captureimage data for particles undergoing deformation. Preferably, themorphology dataset generated is characterized by a high frame rate, suchthat the morphology dataset characterizes multiple stages of deformationfor each particle in the plurality of particles. Furthermore, themorphology dataset is preferably generated in a continuous manner and inreal time; however, the morphology dataset can alternatively begenerated in any other suitable manner.

Block S250 recites: generating a fluorescence dataset characterizingfluorescence of each particle of the plurality of particles within thedeformation region, and functions to generate a dataset that can be usedto extract a set of fluorescence parameters for generation of ananalysis based upon fluorescence parameters. The fluorescence dataset ispreferably generated in Block S250 using an embodiment of the detectionmodule and fluorescence subsystem described above; however, thefluorescence dataset can additionally or alternatively be generatedusing any suitable module including a photodetector configured to detectlight emitted by fluorescent labels being excited by excitationwavelengths of light. Preferably, the fluorescence dataset is generatedin a continuous manner and in real time; however, the fluorescencedataset can alternatively be generated in any other suitable manner.Furthermore, Block S250 is performed concurrently with Block S240, suchthat the morphology dataset and the fluorescence dataset aresimultaneously or nearly simultaneously generated, and deformationcharacteristics and fluorescence parameters can be temporally matched orotherwise synchronized to each particle in the plurality of particles.

Block S260 recites: outputting an analysis of the plurality of particlesbased at least in part on the morphology dataset and the fluorescentdataset for the plurality of particles S260, and functions to produce ananalysis characterizing the particles of interest based upon multipletypes of parameters (e.g., mechanical, deformation, fluorescence,biochemical, etc.). Block S260 is preferably implemented at anembodiment of the processor described above; however, Block S260 canadditionally or alternatively be performed using any suitable processingelement configured to generate an analysis based upon the morphologydataset and the fluorescence dataset. In variations, Block S260 can thusbe implemented at a processor including a first module that extracts theset of deformation characteristics from the morphology dataset; a secondmodule that extracts the set of fluorescence parameters from thefluorescence dataset; a fourth module configured to synchronize themorphology dataset and the fluorescence dataset based upon a deformationcharacteristic and a fluorescence parameter; and a fourth moduleconfigured to generate the analysis. As such, Block S260 can furtherinclude, as illustrated in FIG. 16: extracting a set of deformationcharacteristics from the morphology dataset S261; extracting a set offluorescence parameters from the fluorescence dataset S262; andtemporally synchronizing the morphology dataset and the fluorescencedataset based upon a deformation characteristic and a fluorescenceparameter S263, as shown in FIG. 16.

In Blocks S260 and S261, the set of deformation characteristicspreferably provide morphological characteristics indicative ofphenotype, such as nuclear size, chromatin decondensation, cytoskeletaldisassembly/fluidization, and membrane compromise/lysis. The set ofdeformation characteristics can thus include any one or more of:particle deformability, particle circularity, particle size (e.g.,volume, area, etc.), particle asymmetry, and any other suitablemorphological characteristic. In relation to Blocks S260 and S261 inFIG. 16, the method can additionally comprise extracting baselinemorphological particle characteristics S264, including one or more of:initial particle volume, initial particle diameter, initial particleasymmetry, and any other suitable baseline characteristic. Extractingparticle characteristics in Block S264 can be performed as in U.S. Pub.No. 2013/0177935, entitled “Method and Device for High Throughput CellDeformability Measurements”, or in any other suitable manner. Invariations of the method 200 including Blocks S260, S261, and S264, thebaseline characteristics can be used to normalize the set of deformationcharacteristics for each particle in the plurality of particles, and/orcan be used to generate the analysis in any other suitable manner.

In Blocks S260 and S262, the set of fluorescence parameters preferablyprovide characteristics indicative of biomolecular phenotype and caninclude any one or more of: an intensity of emitted light (e.g., averageintensity, peak intensity), a wavelength of emitted light, kineticparameters of fluorescence, and any other suitable fluorescenceparameter. Similar to variations of the method 200 including Block S264,the method 200 can also include extracting baseline fluorescenceparameters S265 (i.e., prior to particle deformation), including one ormore of: initial intensity (e.g., initial average or peak intensity),initial emitted wavelength prior to deformation, initial kineticparameter(s) prior to deformation, and any other suitable baselineparameter. The baseline parameters can be used to normalize the set offluorescence parameters for each particle in the plurality of particles,and/or can be used to generate the analysis in any other suitablemanner.

In Blocks S260 and S263, synchronizing the morphology dataset and thefluorescence dataset can include conditioning at least one of themorphology dataset and the fluorescence dataset S266, whereinconditioning comprises at least one of noise removal by filtering andpeak-finding. Block S266 can include applying signal filters to removesignal noise and applying a peak-finding algorithm to identify atime-dependent sequence of particles. Block S263 can further includeimplementing a sequence matching algorithm S267, an example of which isshown in FIG. 14, to align event vs. time signals of the morphologydataset with event vs. time signals of the fluorescence dataset. Invariations, Block S263 can further include synchronizing the morphologydataset and the fluorescence dataset based upon data generated fromcalibration particles of the sample volume S268. In examples, thecalibration particles can comprise rigid fluorescent calibrationmicrospheres characterized by identifiable deformability andfluorescence signatures can be used to synchronize the morphologydataset with the fluorescence dataset, irrespective of time stamps. Instill other variations, relationships between deformability (or anyother suitable deformation characteristic) and emitted fluorescenceintensity can be used to synchronize the sets of data in Block S263.However, the morphology dataset and the fluorescence dataset can besynchronized in any other suitable manner.

The analysis generated in Block S260 can comprise a correlation betweenmechanical and biochemical/biomolecular markers for the particles ofinterest, which can be used to identify mechanical (e.g., deformation)characteristics, fluorescence parameters, and/or combinations ofmechanical and fluorescence parameters useful for characterizingparticles of the plurality of particles. In specific applications, theanalysis generated in Block S260 can be used identify activation statesof specific cell lines (e.g., blood mononuclear cell activation bymitogens or inflammatory processes, granulocyte activation withcytokines or blood streams infections, as identified by deformabilityand surface expression of activation markers), with importantimplications in label-free monitoring of diseases, diagnosis ofdiseases, treatment of diseases, and prediction of transplant rejection.In additional applications, the analysis generated can be used toidentify phenotypic connections between stem cells and cancers (e.g.,Jurkat and HL60), used to identify differentiation indicators for stemcells, and used for identification of cells within a diverse populationsof cells within diverse populations of cells in body fluid samples fromhealthy or diseased patients (e.g., resting or activated leuokocytes,PBMCs, and granulocytes as in blood, or pleural fluid). As such, BlockS260 can be further include aggregating a library of data of multipletypes of phenotypic markers based upon the analysis S269, wherein thelibrary characterizes phenotypic markers (e.g., mechanical, deformation,fluorescence, etc.) for a variety of biological particles, using ahigh-throughput approach.

Generating an analysis in Block S260 can thus comprise conducting astatistical analysis (e.g., correlation, t-test, ANOVA, etc.) toinvestigate relationships between deformation and fluorescenceparameters. Additionally or alternatively, Block S260 can includegenerating a classification and regression tree (CART) to enhanceidentification, and can further include using a receiver operatingcharacteristic (ROC) curves to assess correct identification ofparticles Furthermore, linear discriminate analyses can be used in BlockS260 to identify similarities and/or differences between differentsample volumes, which can be used, for example, to stratify samples fromdifferent patients.

As shown in FIG. 15, the method can further comprise Block S270, whichrecites: storing at least one of the morphology dataset, thefluorescence dataset, and the analysis. Block S270 functions to receivedata related to deformation characteristics of the plurality ofparticles, fluorescence parameters of the plurality of particles, andcorrelations between deformation and fluorescence parameters for eachparticle in the plurality of particles. Block S270 can additionallyfunction to store system parameters used to generate the datasets and/orthe analyses, and can further function to enable data transmission to auser or another entity involved with the analysis. Block S270 ispreferably implemented using an embodiment of the storage moduledescribed above; however, Block S270 can be implementing using any othersuitable storage module.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

The invention claimed is:
 1. A system for deforming and analyzing aplurality of cells carried in a sample volume, the system comprising: asubstrate defining an inlet, configured to receive the sample volume,and an outlet; a fluidic pathway fluidly coupled to the inlet and theoutlet and defining a delivery region located upstream of a deformationregion configured to deform one or more cells in the plurality of cells,wherein the fluidic pathway includes a first branch configured todeliver a first portion of the sample volume in a first flow, and asecond branch configured to deliver a second portion of the samplevolume in a second flow that opposes the first flow, wherein anintersection of the first flow and the second flow defines thedeformation region, and wherein the delivery region is fluidicallycoupled with at least one of the first and the second branches; animaging subsystem comprising a first light source configured to transmitlight at a first wavelength range through a portion of the fluidicpathway that includes the deformation region and into an objective lens,the objective lens configured to magnify light from the deformationregion onto a high speed camera having a frame rate of at least 10,000frames per second for generating a morphology dataset of one or morecells in the plurality of cells, a fluorescence subsystem comprising asecond light source configured to emit light at a second wavelengthrange at the same time light is transmitted at the first wavelengthrange through the portion of fluidic pathway that includes thedeformation region by the first light source of the imaging subsystemand focus light onto a portion of the fluidic pathway that is locatedeither upstream or downstream of the deformation region, in which thefocused light produces emitted light at a third wavelength range fromfluorescent labels on the plurality of cells that is collected by theobjective lens and focused onto one or more photodetectors forgenerating the fluorescence dataset characterizing fluorescence of oneor more cells in the plurality of cells; a dichroic mirror interposed inan optical path between the one or more photodetectors and the objectivelens and also interposed in another optical path between the camera andthe objective lens, wherein the dichroic mirror is configured to reflectand redirect light originating from one of the first light source andthe emitted light from the fluorescent labels at the third wavelengthrange, and to transmit light from the other of the first light sourceand the emitted light from the fluorescent labels; and a processorconfigured to output a phenotype analysis of the plurality of cellsbased on the morphology dataset and the fluorescence dataset for theplurality of cells.
 2. The system of claim 1, wherein the deliveryregion is fluidically coupled to the inlet at one end and coupled toboth the first branch and the second branch and is configured to directsubstantially all of the plurality of cells into the first branch, andwherein the second branch containing the second portion of the sample issubstantially free of cells.
 3. The system of claim 1, wherein thedeformation region is further coupled to an external flow inlet, whereinan external flow entering the external flow inlet is configured togenerate an opposing flow at the deformation region.
 4. The system ofclaim 1, wherein the first wavelength range, the second wavelengthrange, and the third wavelength range are substantially non-overlappingwith each other.
 5. The system of claim 1, further comprising a firstmodule configured to extract a set of deformation characteristics fromthe morphology dataset, a second module configured to extract a set offluorescence parameters from the fluorescence dataset, and a thirdmodule configured to temporally synchronize for each single cell passingthrough the deformation region the morphology dataset and thefluorescence dataset based upon a deformation characteristic and afluorescence parameter, wherein the first, second, and third modules areconfigured to be executed by the processor.
 6. The system of claim 5,wherein the third module is configured to synchronize the morphologydataset and the fluorescence dataset based upon substantiallysimultaneous electronic triggering of the imaging subsystem and thefluorescence subsystem.
 7. The system of claim 1, further comprising apump configured to fluidly couple to the inlet, wherein the pump and thedelivery region cooperate to deliver the plurality of cells, in singlefile and at substantially uniform velocity, into the deformation region.8. The system of claim 7, wherein the delivery region comprises a sheathfluid inlet configured to receive a sheath fluid that hydrodynamicallyfocuses the plurality of cells prior to entering the deformation region.9. The system of claim 7, wherein the delivery region is configured todirect the plurality of cells into the deformation region based uponinertial focusing.
 10. The system of claim 9, wherein the deliveryregion comprises a channel having a width and a height dimension,wherein the width dimension is larger than the height dimension, andhaving a plurality of serially arranged constrictions oriented along theheight dimension.
 11. A method for deforming and analyzing a pluralityof cells carried in a sample volume, the method comprising: receivingthe sample volume comprising the plurality of cells; diverting a firstportion of the sample volume in a first flow and a second portion of thesample volume in a second flow, substantially opposed to the first flow,such that an intersection of the first and the second flows defines adeformation region; delivering the plurality of cells into thedeformation region; illuminating one or more cells of the plurality ofcells within the deformation region using a first light source andilluminating one or more cells of the plurality upstream or downstreamof the deformation region using a second light source of a differentwavelength range substantially non-overlapping in wavelength with thefirst light source; generating a morphology dataset characterizingdeformation and morphology of the one or more cells of the plurality ofcells upstream or within the deformation region using the first lightsource and generating a fluorescence dataset characterizing fluorescenceof one or more cells of the plurality of cells upstream or within thedeformation region using the second light source, wherein the morphologydataset and the fluorescence dataset are synchronized as to each cellwithin the plurality of cells, wherein the morphology dataset isdetected with a high speed camera having a frame rate of at least 10,000frames per second and the fluorescence dataset is detected with one ormore photodetectors; and outputting a phenotype analysis of theplurality of cells based on the morphology dataset and the fluorescencedataset for the plurality of cells.
 12. The method of claim 11, furthercomprising filtering the sample volume in at least one of the first flowand the second flow, wherein filtering comprises separating out theplurality of cells based upon size.
 13. The method of claim 11, furthercomprising focusing the cells prior to entry into the deformation regionbased upon inertial focusing.
 14. The method of claim 11, furthercomprising generating a set of deformation characteristics from themorphology dataset, wherein the set of deformation characteristicscomprises at least one of cell deformability and cell circularity. 15.The method of claim 11, further comprising generating a set offluorescence parameters from the fluorescence dataset, wherein the setof fluorescence parameters includes an intensity of emitted light fromfluorescent labels bound to the plurality of cells.
 16. The method ofclaim 11, further comprising displaying at least one of the morphologydataset, the fluorescence dataset, and the phenotype analysis, andgenerating a rendering, based upon the analysis, at a user interface.17. A system for deforming and analyzing a plurality of particlescarried in a sample volume, the system comprising: a substrate definingan inlet and an outlet and a fluidic pathway interposed there between; afocusing region disposed in the fluidic pathway and coupled to the inletat one end and at a downstream end thereof to a trifurcation comprisinga central branch channel, a first siphoning branch channel, and a secondsiphoning branch channel; a deformation region disposed downstream ofthe trifurcation comprising an intersection formed between the centralbranch channel, the first siphoning branch channel, and the secondsiphoning branch channel, wherein the first siphoning branch channel andthe second siphoning branch channel intersect with the central branchchannel in a substantially orthogonal orientation and wherein thecentral branch channel contains the plurality of particles and firstsiphoning branch channel and the second siphoning branch channel aresubstantially free of particles; a detection module comprising a cameraconfigured to generate a morphology dataset characterizing deformationof one or more particles in the plurality of particles, and comprisingone or more photodetectors configured to generate a fluorescence datasetcharacterizing fluorescence of one or more particles in the plurality ofparticles; and a processor configured to output a phenotype analysis ofthe plurality of particles based on the deformation dataset and thefluorescence dataset for the plurality of particles.